Method for selection of insertion mutations

ABSTRACT

The present invention provides a highly-efficient method for the selection and identification of insertional mutants. In the technique, a non-selective amplification is used to isolate a plurality of insertion events from a population of individuals comprising insertion mutations. Specific insertion events can then be identified from the population by the use of gene specific probes or primers. Through the identification of mutants for a particular gene, data may be obtained regarding the function and phenotypic effects of that gene, and thereby, the gene can be employed in the creation of novel biotechnological products.

The present application is a continuing application of U.S. Ser. No.08/932,280, filed Sep. 17, 1997 now U.S. Pat. No. 6,013,486 Jan. 11,2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of molecularbiology. More particularly, it concerns methods and compositions for theidentification and selection of insertional mutants.

2. Description of Related Art

Mutants are powerful tools in the investigation of physiological,developmental, and cell biological processes. Starting with a phenotypicmutant generated by chemical mutagenesis, it is possible to use agenetic map-based strategy to clone a gene (Arondel et al., 1992).Mutations derived from insertional mutagenesis are particularly usefulin that they provide “tagged” copies of the mutated gene which mayreadily be cloned (Yanofsky et al., 1990). However, molecular genetictechniques have advanced such that today most genes are cloned andsequenced long before their function is characterized genetically(Newman et al., 1994). For many genes, phenotypic screens are notavailable, and mutations which cause lethality remain undetectable. Whathas been missing is a simple and reliable strategy to go from a gene orprotein sequence to the identification of specific mutants.

One solution to problems associated with mutant identification was touse the polymerase chain reaction (PCR) to screen for P-elementmutations in sequenced genes of Drosophila (Ballinger et al., 1989;Kaiser et al., 1990). This approach also enhanced the genetics ofCaenorhabditis (Rushforth et al., 1993; Zwaal et al., 1993), wheretransposable element mutations are now commonly isolated for known genesequences. In these systems, transposon-induced mutations are isolatedfor known gene sequences by the general strategy known as“site-selected” mutagenesis. Basically, the method relies on the powerof PCR to amplify a collection of specific junction fragments between aninserted element and a known target gene sequence from large pools ofrandomly inserted elements. One primer is used which is homologous tothe end of the inserted element with its 3′ end facing outward and oneprimer within the target gene is used to amplify the sequences at thejunction of the insertion. In plants, similar approaches have been usedto identify insertion mutations in Petunia, using the transposon dTph1(Koes et al. 1995), and in Arabidopsis using collections of T-DNAtransformed lines (Krysan et al., 1996; Mckinney et al., 1995). InKrysan et al. (1996), 9100 independent T-DNA-transformed Arabidopsislines (averaging 1.4 insertions per genome) were subjected tosite-selected mutagenesis and 17 T-DNA insertions within 63 genes wereidentified.

While techniques based on the gene-specific amplification of insertionaljunctions have been useful in the isolation of a number of mutants, theyhave had limited success in applications toward large-scale genomicinvestigations. The need for individual amplifications of each genebeing investigated represents a significant hindrance when seeking toidentify more than a small number of insertional mutants. There is,therefore, a great need in the art for a method by which large numbersof insertional mutants may be rapidly and efficiently identified.

SUMMARY OF THE INVENTION

The present invention seeks to overcome deficiencies in the prior art byproviding a highly efficient method for selecting insertion events.Therefore, one apsect of the current invention is a method foridentifying an insertion event in a genome comprising the steps of: (a)preparing a first DNA composition enhanced for a plurality of insertionjunctions; (b) preparing at least a first detectable array including thefirst DNA composition; and (c) detecting the insertion event from thefirst array. The step of preparing a first DNA composition may compriseamplification of insertion junctions with inverse PCR, vectorette PCR,primer-adapted PCR, AIMS or any other suitable procedure. The method canfurther comprise preparing at least a second DNA composition, andadditionally any greater number of DNA compositions desired by the userof the invention. The additional DNA compositions may be prepared on thesame, or other arrays, as desired by the user of the invention.

In another aspect of the invention, the detectable array can comprisethe first and second DNA compositions arranged on a solid support. Thesolid support can be a microscope slide, and the insertion event can bedetected by hybridization with a fluorescently labeled probe comprisingcloned DNA, and/or be detected by hybridization with a probe labeledwith an antigen, where the antigen is detected with a molecule whichbinds the antigen. Alternatively, the insertion event can be detected byPCR. In another embodiment of the invention, the array has a solidsupport comprising a nitrocellulose filter, and the insertion event canbe detected by hybridization with a radioactively-labeled probecomprising cloned DNA. The method of detecting can further comprisehybridization of a gene-specific probe to the array. In particularembodiments, the DNA compositions of the array will comprise DNA whichhas been pooled from multiple individuals. The DNA in the compositionscan be derived from potentially any species, including DNA from plants,animals, prokaryotes and lower eukaryotes. In particular embodiments,the DNA may be from a monocot plant, and may further defined as frommaize, rice, wheat, barley, sorghum, oat, or sugarcane. In otherembodiments, the monocot DNA is maize DNA. The plant DNA may also bedicot DNA, and may be derived from a species selected from the groupconsisting of cotton, tobacco, tomato, soybean, sunflower, oil seed rape(canola), alfalfa, potato, strawberry, onion, broccoli, Arabidopsis,pepper, and citrus. In particular embodiments of the invention the dicotplant DNA is Arabidopsis thaliana DNA. In still other embodiments theDNA is animal DNA.

Still yet another aspect of the invention provides a method ofdetermining the function of a DNA sequence. In particular embodiments ofthe invention the method comprises the steps of: (a) amplifying aplurality of insertion junctions from a DNA composition comprisinginsertion mutations; (b) creating at least a first array comprising saidinsertion junctions; (c) detecting at least a first mutation in said DNAsequence from said array using a primer or probe specific to said DNAsequence; and (d) determining the function of said DNA sequence bycomparing individuals comprising said mutation in said DNA sequence tocorresponding individuals lacking said mutation in said DNA sequence. Inthe method, the DNA composition may comprise plant DNA. In particularembodiments the plant DNA may be further defined as monocot plant DNA,and may be still further defined as derived from a species selected fromthe group consisting of maize, rice, wheat, barley, sorghum, oat, andsugarcane. In particular embodiments, the monocot DNA comprises maizeDNA. The plant DNA can also comprise dicot plant DNA, and may be stillfurther defined as derived from a species selected from the groupconsisting of cotton, tobacco, tomato, soybean, sunflower, oil seed rape(canola), alfalfa, potato, strawberry, onion, broccoli, Arabidopsis,pepper, and citrus. In particular embodiments, the DNA composition isArabidopsis thaliana DNA.

Still yet another aspect of the invention provides a method forisolating a plant comprising a desired integration event. In particularembodiments of the invention, the method comprises the steps of: (a)integratively transforming a plurality of plants; (b) obtaining DNA fromsaid plants; (c) amplifying a plurality of transgene insertion junctionsfrom said DNA; (d) preparing at least a first array comprising saidamplified insertion junctions; and (e) detecting a desired integrationevent with a probe or primer corresponding a preselected genomic region.In particular embodiments, the plant may be further defined as a monocotplant, and may be still further defined as derived from a speciesselected from the group consisting of maize, rice, wheat, barley,sorghum, oat, and sugarcane. In other embodiments, the monocot plant isa maize plant. The plant can also comprise a dicot plant, and may bestill further defined as a species selected from the group consisting ofcotton, tobacco, tomato, soybean, sunflower, oil seed rape (canola),alfalfa, potato, strawberry, onion, broccoli, Arabidopsis, pepper, andcitrus. In particular embodiments, the plant is an Arabidopsis thalianaplant.

Still yet another aspect of the invention provides a plant preparable bya process comprising the steps of: (a) integratively transforming aplurality of plants; (b) obtaining DNA from said plants; (c) amplifyinga plurality of transgene insertion junctions from said DNA; (d)preparing at least a first array comprising said amplification insertionjunctions; and (e) detecting a plant having a desired transgeneinsertion event using a probe or primer corresponding to the selectedgenomic region. The plant may be further defined as a monocot plant,wherein the monocot plant may be still further defined as a monocotplant selected from the group consisting of maize, rice, wheat, barley,sorghum, oat, and sugarcane. In particular embodiments the monocot plantis maize. The plant may also be a dicot plant, and in particularembodiments, still further defined as selected from the group consistingof cotton, tobacco, tomato, soybean, sunflower, oil seed rape (canola),alfalfa, potato, strawberry, onion, broccoli, Arabidopsis, pepper, andcitrus. In particular embodiments the dicot plant is an Arabidopsisthaliana plant.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention represents a significant advance over priormethods for identifying insertional mutations in that it allows for thesimultaneous screening of large numbers of unique insertion events.Therefore, the first step of the invention, in one embodiment, willinvolve obtaining or generating a population of individuals withinsertional mutations from which to screen for the mutant of interest. Apreferred population will represent a large number of insertionalmutations such that there will be a high probability of identifying amutant for any given locus within the population. In a preferredembodiment, the next step will generally involve isolating DNA from thepopulation of insertional mutations and creating pools which contain DNAfrom various different combinations of individuals. The pools aredesigned such that, through analysis of multiple pools, sequencesrepresenting single members of a population can be identified withoutthe need for individual analysis of each member of the population. Theinsertion junctions present in each pool are then amplifiednon-selectively, providing a broad class of “tagged” insertion junctionswhich can subsequently be detected by use of gene-specific probes orprimers. An efficient means employed for the detection of amplifiedinsertion junctions in the pools is the preparation of arrays arrangedon a suitable solid support material. The labeled gene-specific probesmay then be hybridized and detected directly on the arrays, allowingsimultaneous screening of a large number of pools and ultimateidentification of one or more insertional mutants.

The probability of successfully identifying a chosen insertional mutantwith the current invention will be greatly influenced by thecharacteristics of the starting population(s) from which insertionalmutants will be screened. One important characteristic of the populationwill be the number of insertional mutations it contains. It will, ofcourse, be preferred that any such population contain a sufficientnumber of insertion events that there is a reasonable likelihood ofdetecting at least one insertional mutant from any particular gene orlocus. As such, the mechanism by which insertional mutations aregenerated will be important to the degree of ease with which the currentinvention may be used. While insertion mutations caused by potentiallyany known sequence long enough to be amplified may be detected with thecurrent invention, certain types of insertions will offer advantages.Preferred insertion mutations will be predominately or completelyrandomly distributed throughout the target genome. This will decreasethe likelihood that a particular locus is lacking an insertion mutationin the generated population and also reduce the size of the populationneeded to have a reasonable probability of detecting any given insertionmutation. Also preferred will be insertional mutagens which are capableof producing large numbers of mutations both within individuals andwithin populations, thereby increasing the effective number of mutationswhich may be obtained and subsequently screened. The insertion mutationscreated will also preferably alter gene expression for the mutated genecopy, allowing studies to elucidate the mutated genes' phenotypic effectand function, and potentially creating valuable new phenotypes.

Examples of types of insertion mutations which are contemplated to be ofparticular utility with the current invention will be those created bytransposable elements and transgenes introduced by transformation. Whichtype of these, or another, insertion mutations is utilized with thecurrent invention will typically depend on factors including theorganism being studied, available resources, and the goal of the study.For example, in many dicot plants, transformation with the T-DNA ofAgrobacterium may be readily achieved and large numbers of transformantscan be rapidly obtained. In some monocot plants, however, transformationis less efficient and requires tissue culture steps which arecomparatively time- and labor-intensive, making transformation a muchless attractive alternative. Also, some species have lines with activetransposable elements which can efficiently be used for the generationof large numbers of insertion mutations, while some other species lacksuch options. In particular instances, it may be advantageous to screenmultiple types of insertion mutations, thereby increasing the chance ofdetecting any given desired mutant. Therefore, a number of factors willbe taken into account when choosing the type(s) of insertion mutation tobe identified with the current invention. These factors will be readilyapparent to those of skill in the art in light of the present disclosureand will dependent on the specific goals of the investigation.

(i) Target Organism for Use with the Invention

The current invention is applicable to any species for which insertionalmutants may be obtained. As such, it is specifically contemplated by theinventor that one may wish to use the current invention for theidentification of specific insertion events from plants, animals, lowereukaryotes and prokaryotes. Examples of some animals for which thecurrent invention may be used include poultry, dairy and beef cattle,primates, rodents, swine and insects. Examples of plants which arespecifically contemplated for use with the current invention includemonocots such as maize, rice, wheat, barley, sorghum, oat, andsugarcane, as well as dicots such as cotton, tobacco, tomato, soybean,sunflower, oil seed rape (canola), alfalfa, potato, strawberry, onion,broccoli, Arabidopsis, pepper, and citrus. Maize and Arabidopsisrepresent target plant species which will be particularly advantageousfor use with the current invention.

(ii) Utilization of Transposon-Generated Insertion Mutations

Transposable-elements are an extremely versatile class of insertionalmutagen in that a great variety of transposable elements have beenidentified, with representative elements having been found in alleukaryotic genomes examined (Flavell et al., 1992).

As used herein, the term “transposable element” will mean any mobilegenetic element which is capable of replicative or non-replicativetransposition within a genome, causing insertional mutagenesis at thesite of insertion. One example of a transposable element of maizecontemplated to have particular utility in the generation of insertionmutations is the Mutator element (Bennetzen, 1984; Talbert et al., 1989;see Genbank Accession Numbers: x14224, x14225, g22495, g22466, g22373,m76978 and x97569). Other examples of transposable elements which aredeemed particularly useful insertional mutagens are the Ac element(Geiser et al., 1982; U.S. Pat. No. 4,732,856, specifically incorporatedherein by reference in its entirety) and the tobacco element slide-124(Grappin et al, 1996; Genbank Accession Number x97569).

(iii) Generation of Insertionally Mutagenized Plant Cells byTransformation

There are many methods for transforming DNA segments into cells, but notall are suitable for delivering DNA to plant cells. Suitable methods arebelieved to include virtually any method by which DNA can be introducedinto a cell, such as by Agrobacterium infection (described in, forexample, U.S. Pat. No. 5,591,616, specifically incorporated herein byreference in its entirety); direct delivery of DNA such as byPEG-mediated transformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), byelectroporation (U.S. Pat. No. 5,384,253), by agitation with siliconcarbide fibers (Kaeppler et al. 1990), and by acceleration of DNA coatedparticles (U.S. Pat. No. 5,550,318), etc. Through the application oftechniques such as these, certain cells from virtually any plant speciesmay be stably transformed, and these cells developed into transgenicplants. In certain embodiments, acceleration methods are preferred andinclude, for example, microprojectile bombardment and the like.

One type of insertional mutations which will be of particular use in thecurrent invention are those caused by the T-DNA of Agrobacterium. Animportant advantage of T-DNA-based insertions is that they areapparently randomly distributed in any given genome (reviewed byTinland, 1996). This has been confirmed in Arabidopsis, where a uniformdistribution at the chromosomal level and a random distribution withintranslated and untranslated regions of genes was shown (Aspiroz-Leehanand Feldman, 1997). Moreover, sequence analysis of target sites showsthat: (i) integration is not site-specific; (ii) T-DNA integration canlead to small deletions (13-72 bp) at the site of insertion; and (iii)the left-end border of integrated T-DNA is usually poorly conserved ascompared to the right border sequences, which can be conserved up to thenucleotide that is covalently attached to the VirD2 movement protein(Tinland, 1996). Additionally, one or more T-DNA loci (chromosomalintegration sites) can frequently be found integrated into the genome ofa plant cell, and the same cell can carry T-DNAs derived from differentAgrobacteria cells (DeBlock et al., 1991; Depicker, 1995). Frequently,the structure of the T-DNA at a locus can be complex, involving theintegration of direct and inverted T-DNA repeats.

1. Electroporation

Where one wishes to introduce DNA by means of electroporation, it iscontemplated that the method of Krzyzek et al. (U.S. Pat. No. 5,384,253,incorporated herein by reference in its entirety) will be particularlyadvantageous. In this method, certain cell wall-degrading enzymes, suchas pectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells. Alternatively, recipient cells are made moresusceptible to transformation, by mechanical wounding.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus, or alternatively one may transform immature embryos or otherorganized tissue directly. One would partially degrade the cell walls ofthe chosen cells by exposing them to pectin-degrading enzymes(pectolyases) or mechanically wounding in a controlled manner. Suchcells would then be recipient to DNA transfer by electroporation, whichmay be carried out at this stage, and transformed cells then identifiedby a suitable selection or screening protocol, dependent on the natureof the newly incorporated DNA.

2. Microprojectile Bombardment

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment (U.S. Pat. Nos. 5,550,318;5,538,880; 5,610,042; and PCT Patent Publication No. 94/09699; eachspecifically incorporated herein by reference in its entirety). In thismethod, particles may be coated with nucleic acids and delivered intocells by a propelling force. Exemplary particles include those comprisedof tungsten, gold, platinum, and the like. It is contemplated that insome instances DNA precipitation onto metal particles would not benecessary for DNA delivery to a recipient cell using microprojectilebombardment. However, it is contemplated that particles may contain DNArather than be coated with DNA. Hence, it is proposed that DNA-coatedparticles may increase the level of DNA delivery via particlebombardment but are not, in and of themselves, necessary.

An advantage of microprojectile bombardment, in addition to its being aneffective means of reproducibly stably transforming monocots, is thatneither the isolation of protoplasts (Christou et al., 1988) nor thesusceptibility to Agrobacterium infection is required. An illustrativeembodiment of a method for delivering DNA into maize cells byacceleration is the Biolistics Particle Delivery System, which can beused to propel particles coated with DNA or cells through a screen, suchas a stainless steel or Nytex screen, onto a filter surface covered withmonocot plant cells cultured in suspension. The screen disperses theparticles so that they are not delivered to the recipient cells in largeaggregates. It is believed that a screen intervening between theprojectile apparatus and the cells to be bombarded reduces the size ofprojectiles aggregate and may contribute to a higher frequency oftransformation by reducing the damage inflicted on the recipient cellsby projectiles that are too large. Examples of species for which theBiolistics Particle Delivery System has been successfully used fortransformation include monocot species such as maize, barley, wheat,rice, and sorghum, as well as various dicot species, including tobacco,soybean, cotton, sunflower, and tomato.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate. If desired, one or more screens may be positionedbetween the acceleration device and the cells to be bombarded.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in the manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust variousbombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters.such as gap distance, flight distance, tissue distance, helium pressure,and microprojectile particle size. One may also minimize the traumareduction factors (TRFs) by modifying conditions which influence thephysiological state of the recipient cells and which may thereforeinfluence transformation and integration efficiencies. For example, theosmotic state, tissue hydration, and the subculture stage or cell cycleof the recipient cells may be adjusted for optimum transformation.Results from such small scale optimization studies are disclosed herein,and the execution of other routine adjustments will be known to those ofskill in the art in light of the present disclosure (see, for example,PCT Patent Publication No. 94/09699, specifically incorporated herein byreference in its entirety).

3. Agrobacterium-Mediated Transfer

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., 1983; Rogers et al., 1987). Further,the integration of the T-DNA is a relatively precise process resultingin few rearrangements. The region of DNA to be transferred is defined bythe border sequences, and the intervening DNA is usually inserted intothe plant genome as described (Spielmann et al., 1986; Jorgensen et al.,1987).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described(Rogers et al., 1987) have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Inaddition, Agrobacterium containing both armed and disarmed Ti genes canbe used for the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer. Anexample of one T-DNA which will be especially useful with the currentinvention will be that of SEQ. ID NO. 1.

Agrobacterium-mediated transformation of leaf disks and other tissues,such as cotyledons and hypocotyls, appears to be limited to plants thatAgrobacterium naturally infects. Agrobacterium-mediated transformationis most efficient in dicotyledonous plants and is the preferable methodfor transformation of dicots, including Arabidopsis, tobacco, tomato,and potato. Few monocots appear to be natural hosts for Agrobacterium,although transgenic plants have been produced in asparagus usingAgrobacterium vectors as described (Bytebier et al., 1987). Therefore,commercially important cereal grains, such as rice, corn, and wheat mustusually be transformed using alternative methods. Agrobacterium-mediatedtransformation of maize and rice has, however, been described in U.S.Pat. No. 5,591,616, specifically incorporated herein by reference in itsentirety.

One efficient means by which Agrobacterium plant transformation can bemediated is by way of vacuum infiltration. This procedure is based onthe vacuum infiltration of a suspension of Agrobacterium cellscontaining a binary T-DNA vector into plant tissue, such as, forexample, from Arabidopsis plants. Exemplary procedures for vacuuminfiltration are known to those of skill in the art and are disclosed inBechtold and Bouchez (1995); and Bechtold et al. (1993), each of whichis specifically incorporated herein by reference in its entirety.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single transgene or a few copies of a transgene onone chromosome. Such transgenic plants can be referred to as beinghemizygous. For detection of an insertional mutagen, such a plant may bepreferred, in that many of the mutations may be recessive lethals. Wherethe mutation is not a recessive lethal, a preferred plant may behomozygous for the added structural gene, i.e., a transgenic plant thatcontains two added genes, one gene at the same locus on each chromosomeof a chromosome pair. A homozygous transgenic plant can be obtained bysexually mating (selfing) a hemizygous transgenic plant that contains asingle added gene, germinating some of the seed produced, and analyzingthe resulting plants.

It is to be understood that two different transgenic plants can also bemated to produced offspring that contain multiple,independently-segregating added, insertion events. Specificallycontemplated by the inventor, is the creation of plants which contain 1,2, 3, 4, 5, or even more independently-segregating added insertionevents. Selfing of appropriate progeny can produce plants that arehomozygous for all added insertion mutations. Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated.

4. Other Transformation Methods

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1986; Uchimiyaet al., 1986; Callis et al., 1987; Marcotte et al., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastsare described (Fujimara et al., 1985; Toriyama et al., 1986; Yamada etal., 1986; Abdullah et al., 1986; Omirulleh et al., and 1993 U.S. Pat.No. 5,508,184; each specifically incorporated herein by reference in itsentirety).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, 1989). Also,pollen-mediated transformation may be used (U.S. Pat. No. 5,629,183;specifically incorporated herein by reference) In addition, “particlegun” or high-velocity microprojectile technology can be utilized (Vasil,1992).

Using that latter technology, DNA is carried through the cell wall andinto the cytoplasm on the surface of small metal particles as described(Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988). The metalparticles penetrate through several layers of cells and thus allow thetransformation of cells within tissue explants.

(iv) Generation of Insertionally Mutagenized Animal Cells byTransformation

In certain embodiments of the invention, animal cells comprising novelinsertional mutants may be created by integrative transformation ofrecipient animal cells. Through such methods, which are well known tothose of skill in the art, and others set forth herein, insertionalmutants may be created for virtually any animal, plant, prokaryote orlower eukaryote. Specific methods contemplated by the inventor to be ofuse in the creation of insertional mutants are disclosed herein.

An example of a method of DNA delivery to recipient cells which may beused is viral infection, where a particular construct is encapsulated inan infectious viral particle. For use herein, the virus will be onewhich directs integrative transformation of the transformed cell.Non-viral methods for the transfer of foreign DNA into recipient cellsalso are contemplated in the present invention. In one embodiment of thepresent invention, the construct may consist only of naked DNA orplasmids; however, almost any DNA segment which is capable ofinsertionally mutating a target locus and which has a known sequence maypotentially be used with the current invention. Transfer of the DNA maybe performed by any of the methods mentioned which physically orchemically permeabilize the cell membrane.

1. Liposome-Mediated Transfection

Foreign DNA may be delivered to cells by way of liposomes. Liposomes arevesicular structures characterized by a phospholipid bilayer membraneand an inner aqueous medium. Multilamellar liposomes have multiple lipidlayers separated by aqueous medium. They form spontaneously whenphospholipids are suspended in an excess of aqueous solution. The lipidcomponents undergo self-rearrangement before the formation of closedstructures and entrap water and dissolved solutes between the lipidbilayers (Ghosh et al., 1991). It is contemplated that one may wish tocomplex the DNA to be delivered with Lipofectamine (Gibco BRL).

Liposome-mediated nucleic acid delivery of foreign DNA in vitro has beendemonstrated to be a reliable means of transformation (Nicolau et al.,1982; Fraley et al., 1979; Nicolau et al., 1987). Wong et al. (1980)demonstrated the feasibility of liposome-mediated delivery andexpression of foreign DNA in cultured chick embryo, HeLa, and hepatomacells.

In certain embodiments, the liposomes may be complexed with ahemagglutinating virus (HVJ). This has been shown to facilitate fusionwith the cell membrane and promote cell entry of liposome-encapsulatedDNA (Kaneda et al., 1989). In other embodiments, the liposome may becomplexed or employed in conjunction with both HVJ and HMG-1.

2. Electroporation

In certain embodiments of the present invention, insertionallymutagenized animal cells may be created via electroporation.Electroporation involves the exposure of a suspension of cells and DNAto a high-voltage electric discharge. This technique is widelyapplicable to virtually any eukaryotic cell and may also be used fortransformation of prokaryotes.

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre-B lymphocytes have been transfected with humankappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur-Kaspa et al., 1986) in this manner.

3. Calcium Phosphate Precipitation or DEAE-Dextran Treatment

In other embodiments of the present invention, the foreign DNA may beintroduced to the cells using calcium phosphate precipitation. Human KBcells have been transfected with adenovirus DNA (Graham et al., 1973)using this technique. Also in this manner, mouse L(A9), mouse C127, CHO,CV-1, BHK, NIH3T3, and HeLa cells were transfected with a neomycinmarker gene (Chen and Okayama, 1987), and rat hepatocytes weretransfected with a variety of marker genes (Rippe et al., 1990).

In another embodiment, the foreign DNA may be delivered into the cellusing DEAE-dextran followed by polyethylene glycol. In this manner,reporter plasmids were introduced into mouse myeloma and erythroleulemiacells (Gopal, 1985).

4. Direct Microinjection or Sonication Loading

In still further embodiments of the invention, insertionally mutagenizedanimal cells may be created by the delivery of foreign DNA withmicroinjection or sonication loading. Direct microinjection has beenused to introduce nucleic acid constructs into Xenopus oocytes (Harlandand Weintraub, 1985), and LTK⁻ fibroblasts have been transfected withthe thymidine kinase gene by sonication loading (Fechheimer et al.,1987). A similar method involves injecting a polyamino acid/DNA complexinto the cytoplasm of animal cells to effect transformation (U.S. Pat.No. 5,523,222 specifically incorporated herein by reference).

5. Receptor-Mediated Transfection

A still further method for delivery of foreign DNA involves the deliveryof constructs to the target cells with receptor-mediated deliveryvehicles. These take advantage of the selective uptake of macromoleculesby receptor-mediated endocytosis that will be occurring in the targetcells. In view of the cell type-specific distribution of variousreceptors, this delivery method adds another degree of specificity tothe transformation. Specific delivery in the context of anothermammalian cell type is described by Wu and Wu (1993).

Certain receptor-mediated gene targeting vehicles comprise a cellreceptor-specific ligand and a DNA-binding agent. Others comprise a cellreceptor-specific ligand to which the DNA construct to be delivered hasbeen operatively attached. Several ligands have been used forreceptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al., 1990;Perales et al., 1994; European Patent No. 0 273 085), which establishesthe operability of the technique. In the context of the presentinvention, the ligand will be chosen to correspond to a receptorspecifically expressed on the neuroendocrine target cell population.

In other embodiments, the DNA delivery vehicle component of acell-specific gene targeting vehicle may comprise a specific bindingligand in combination with a liposome. The nucleic acids to be deliveredare housed within the liposome and the specific binding ligand isfunctionally incorporated into the liposome membrane. The liposome willthus specifically bind to the receptors of the target cell and deliverthe contents to the cell. Such systems have been shown to be functionalusing systems in which, for example, epidermal growth factor (EGF) isused in the receptor-mediated delivery of a nucleic acid to cells thatexhibit upregulation of the EGF receptor.

In still further embodiments, the DNA delivery vehicle component of thetargeted delivery vehicles may be a liposome itself, which willpreferably comprise one or more lipids or glycoproteins that directcell-specific binding. For example, Nicolau et al. (1987) employedlactosyl-ceramide, a galactose-terminal asialganglioside, incorporatedinto liposomes and observed an increase in the uptake of the insulingene by hepatocytes.

Therefore, transformation of host species may be used in a similarmanner to transposon-tagging. In transposon tagging, as with integrativetransformation, insertion mutations are created in the genomes of targetorganisms by transposable elements. This creates mutant individuals fromwhich mutant phenotypes can be identified. DNA can then be isolated fromthe mutants and used for the creation of genomic libraries. The mutatedgene can then be efficiently cloned through the use the transposon as a“tag”. Typically, a number of candidate genes will first be identified.These may then be confirmed by complementation experiments or DNAsequencing and homology searches for related known genes.

I. Amplification of Insertion Junctions

An important aspect of the current invention is that it allows selectionof specific insertional mutants from a diverse class of insertionevents. For this purpose, one step of the invention utilizes thenon-selective amplification of insertion junctions. As used herein, theterm “non-selective amplification” is used to denote amplificationprocedures which will simultaneously amplify a broad class of insertionjunctions without the need for a single gene-specific primer. Techniqueswhich are contemplated by the inventor as being particularly useful forthe non-specific amplification are inverse PCR, vectorette PCR, andprimer-adapted PCR, with vectorette PCR being most preferred, althoughpotentially any method capable of amplifying a diverse class ofinsertion junctions may be used.

(i) Inverse PCR

Inverse polymerase chain reaction (IPCR) is an extension of thepolymerase chain reaction that permits the amplification of regions thatflank any DNA segment of known sequence, either upstream or downstream(see U.S. Pat. No. 4,994,370, specifically incorporated herein byreference in its entirety). The essence of IPCR is that, bycircularizing a restriction enzyme fragment containing a region of knownsequence plus flanking DNA, PCR can be performed using oligonucleotideswhose sequence is taken from the single region of known sequence andoriented with respect to one another such that their 5′ to 3′ extensionproducts proceed toward each other by going “around the circle” throughwhat originally was flanking DNA. This leads to the amplification of DNAstrands containing what was originally flanking DNA. The advantage of atechnique such as IPCR, with respect to the current invention, is thatusing a single primer set one may amplify a representative sample ofinsertion junctions from a particular group of individuals.

Selection of appropriate restriction enzymes for use in IPCR can bedetermined empirically by Southern blotting and hybridization proceduresusing all or part of the core region. Selection of the appropriatefragment can be facilitated by computer search methods, since in mostcases the entire nucleotide sequence of the core (e.g., wellcharacterized insertional mutagens such as transposable elements ortransgenes) region will be known. The amplified fragment should be nogreater than 2-3 kilobases (kb), which is a limitation imposed by thesize of a region that can be efficiently amplified using the mostcommonly available methods of PCR. However, recently PCR techniques havebeen developed, termed Long PCR, which are capable of amplifying DNAfragments of 20 kb or more.

After restriction enzyme digestion, the DNA fragments produced by therestriction enzyme are diluted and ligated under conditions that favorthe formation of monomeric circles (Collins et al., 1984). The resultingintramolecular ligation products are then used as substrates forenzymatic amplification by PCR using oligonucleotide primers homologousto the ends of the core sequence but facing in opposite orientations.The primary product of the resulting amplification is a lineardouble-stranded molecule including segments situated both 5′ and 3′ tothe core region. The junction between the original upstream anddownstream regions, otherwise ambiguous, can be identified as therestriction site of the restriction enzyme that was used to produce thelinear fragments prior to ligation. By selecting a restriction enzymethat cleaves inside a known core sequence, the IPCR procedure willproduce products containing only the upstream or only the downstreamflanking regions.

(ii) Vectorette PCR

There are three basic steps in the technique of vectorette PCR: (1)digestion of target DNA with one or more suitable restriction enzymes;(2) ligation of suitable synthetic oligonucleotides onto the digestedDNA; and (3) PCR using a specific primer and a primer directed towardthe synthetic oligonucleotides (see European Patent No. 0 439 330,specifically incorporated herein by reference in its entirety). In thisprocedure, nonspecific amplification of all digested fragments isavoided by the design of specific fragments of synthetic DNA, calledvectorettes. Vectorettes are designed so that they can be amplified onlyif they are attached to the DNA insertional mutagen. The vectorette isonly partially double-stranded and contains a central mismatched region.The vectorette PCR primer has the same sequence as the bottom strand ofthis mismatched region and therefore has no complementary sequence toanneal to in the first cycle of PCR. In the first cycle of PCR, only theknown primer, which is directed toward the insertional mutagen, willprime DNA synthesis. This will produce a complementary strand for thevectorette PCR primer to anneal to in the second cycle of PCR. In thesecond and subsequent cycles of PCR, both primers can prime synthesis,with the end result being that the only fragment amplified contains theinsertional mutagen and flanking DNA of the insertion site.

(iii) Primer Adapted PCR

The primer-adapted PCR technique is a derivation of ligation-mediatedsingle-sided PCR (Fors et al., 1990; Mueller et al., 1989). This methoduses linker ligation and subsequent amplifications with a linker-primerand multiple insertional-mutagen-specific primers (“nested” primers) toobtain specificity. The ligation-mediated single-sided PCR protocolinvolves multiple PCRs and subsequent purifications on agarose gels.

The amplification procedure involves, as a first step, restriction withan appropriate restriction enzyme, such as Sau3AI, and ligation ofprimer adapters to the different DNA size fractions. Then, approximately50 cycles of linear amplification are performed using an internalbiotinylated primer complimentary to the insertional mutagen. Thebiotinylated linear PCR product is purified from the rest of the genomicDNA with streptavidin-coated magnetic beads and subjected to exponentialPCR using the adapter-primer and the insertional-mutagen specificprimer. The result of this first round of exponential PCR may bevisualized on an agarose gel and used in the preparation of arrays.Successful, specific amplification should be indicated by a series ofbands on the agarose gel.

In order to avoid the purification steps required because ofnon-specificity in the PCR, an additional step may be introduced thatinvolves linear amplification of the target sequence with a biotinylatedprimer and separation of the product with the aid of streptavidin-coatedmagnetic beads (Hultman et al., 1989; Rosenthal and Jones, 1990). Thisstrategy may be employed in combination with ligation of oligo-cassettesto restricted DNA to directly amplify unknown regions which flank aninsertional mutagen (Rosenthal and Jones, 1990).

The basic concept of the method is to employ an “internal” primercomplementary to a known sequence in the insertional mutagen incombination with an “external” adapter-primer. First, primer adaptersare ligated onto the genomic DNA digested with a suitable enzyme (forexample, Sau3AI), then a linear PCR is performed with the insertionalmutagen-complimentary primer, which is biotinylated. Since the linearPCR product is biotinylated, it can then be purified from the rest ofthe genomic DNA with the aid of streptavidin-coated magnetic beads.After the magnetic purification, an exponential PCR is carried out usingthe internal primer in combination with the adapter-primer. An extraround of PCR with a nested internal primer and the adapter-primer can beperformed to achieve increased specificity. The amplified product canthen be used for the production of arrays for ultimate detection ofinsertional mutants.

(iv) Other Methods

As previously stated, any method which may be used to enrich for adiverse collection of insertion junctions may be used with the currentinvention. An example of one such technique disclosed herein for theenrichment of transposon Mu-tagged sites is Amplification of InsertionMutagenized Sites (AIMS), the procedure for which is outlined below, inExample 5 and described by Souer et al. 1995.

II. Detection of Insertional Mutants from Arrays

One aspect of the current invention which allows for efficient selectionof large numbers of insertional mutants is the creation of arrayscomprising insertion-junction-enriched DNA pools. The precise placementof this pooled DNA into specific arrays allows for the simultaneousscreening of potentially thousands of insertion mutations. The methodinvolves the placement and binding of DNA to known locations, termedsectors, on a solid support. Through hybridization of a desired specificprobe or primer to the array, for example, insertion mutationscorresponding to that gene may be identified from the total collectionof insertional mutants. Further, because the amplification step may beconducted repeatedly, a large number of identical or non-identicalarrays may be produced, thereby allowing simultaneous screening withmany different locus-specific probes or primers.

Many different methods for preparation of arrays of DNA on solidsupports are known to those of skill in the art. Specific methods ofwhich are disclosed in, for example, Affinity Techniques, EnzymePurification: Part B, Meth. Enz. 34 (ed. W. B. Jakoby and M. Wilchek,Acad. Press, N.Y. (1974) and Immobilized Biochemicals and AffinityChromatography, Adv. Exp. Med. Biol. 42 (ed. R. Dunlap, Plenum Press,N.F. 1974), each specifically incorporated herein by reference in itsentirety). Examples of other techniques which have been describedinclude the use of successive application of multiple layers of biotin,avidin, and extenders (U.S. Pat. No. 4,282,287, specificallyincorporated herein by reference in its entirety); through methodsemploying a photochemically active reagent and a. coupling agent whichattaches the photoreagent to the substrate (U.S. Pat. No. 4,542,102,specifically incorporated herein by reference in its entirety), use ofpolyacrylamide supports on which are immobilized oligonucleotides (PCTPatent Publication No. 90/07582, specifically incorporated herein byreference in its entirety), through use of solid supports on whicholigonucleotides are immobilized via a 5′-dithio linkage (PCT PatentPublication No. 91/00868, specifically incorporated herein by referencein its entirety); and through use of a photoactivateable derivative ofbiotin as the agent for immobilizing a biological polymer of interestonto a solid support (see U.S. Pat. No. 5,252,743; and PCT PatentPublication No. 91/07087 to Barrett et al., each specificallyincorporated herein by reference in its entirety). In the case of asolid support made of nitrocellulose or the like, standard techniquesfor UV-crosslinking may be of particular utility (Sambrook et al.,1989).

The solid support surface upon which the array is produced maypotentially be any suitable substance. Examples of materials which maybe used include polymers, plastics, resins, polysaccharides, silica orsilica-based materials, carbon, metals, inorganic glasses, membranes,etc. It may also be advantageous to use a surface which is opticallytransparent, such as flat glass or a thin layer of single-crystalsilicon. Contemplated as being especially useful are nylon filters, suchas Hybond N+ (Amersham Corporation, Amersham, UK). Surfaces on the solidsubstrate will usually, though not always, be composed of the samematerial as the substrate, and the surface may further contain reactivegroups, which could be carboxyl, amino, hydroxyl, or the like.

It is contemplated that one may wish to use a surface which is providedwith a layer of crosslinking groups (U.S. Pat. No. 5,412,087,specifically incorporated herein by reference in its entirety).Crosslinking groups could be selected from any suitable class ofcompounds, for example, aryl acetylenes, ethylene glycol oligomerscontaining 2 to 10 monomer units, diamines, diacids, amino acids, orcombinations thereof. Crosslinking groups can be attached to the surfaceby a variety of methods that will be readily apparent to one of skill inthe art. For example, crosslinking groups may be attached to the surfaceby siloxane bonds formed via reactions of crosslinking groups bearingtrichlorosilyl or trisalkoxy groups with hydroxyl groups on the surfaceof the substrate. The crosslinking groups can be attached in an orderedarray, i.e., as parts of the head groups in a polymerized LangmuirBlodgett film. The linking groups may be attached by a variety ofmethods that are readily apparent to one skilled in the art, forinstance, by esterification or amidation reactions of an activated esterof the linking group with a reactive hydroxyl or amine on the free endof the crosslinking group.

The ultimate goal of producing an array in accordance with currentinvention, will be in screening large numbers of individuals or subsetsof individuals for detection of an insertional mutant. Therefore, oncethe array is produced, the first step will, in a preferred embodiment,involve hybridizing the array with a solution containing a marked(labeled) probe. For detection of a mutation in a specific gene, thiswill typically involve the use of a cloned DNA segment including thatgene sequence as a probe. Following hybridization, the surface is thenwashed free of unbound probe, and the signal corresponding to the probelabel is identified for those regions on the surface where the probe hashigh affinity. Suitable labels for the probe include, but are notlimited to, radiolabels, chromophores, fluorophores, chemiluminescentmoieties, antigens and transition metals. In the case of a fluorescentlabel, detection can be accomplished with a charge-coupled device (CCD),fluorescence microscopy, or laser scanning (U.S. Pat. No. 5,445,934,specifically incorporated herein by reference in its entirety). Whenautoradiography is the detection method used, the marker is aradioactive label, such as ³²P, and the surface is exposed to X-rayfilm, which is developed and read out on a scanner or, alternatively,simply scored manually. With radiolabeled probes, exposure time willtypically range from one hour to several days. Fluorescence detectionusing a fluorophore label, such as fluorescein, attached to the ligandwill usually require shorter exposure times. Alternatively, the presenceof a bound probe may be detected using a variety of other techniques,such as an assay with a labeled enzyme, antibody, or the like. Othertechniques using various marker systems for detecting bound ligand willalso be readily apparent to those skilled in the art.

Detection may, alternatively, be carried out using PCR. In thisinstance, PCR detection may be carried out in situ on the slide. In thiscase one may wish to utilize one or more labeled nucleotides in the PCRmix to produce a detectable signal. Detection may also be carried out ina standard PCR reaction on the prepared samples to be screened. For thistype of detection, the sectors in the array will not consist of DNAbound to solid support but will consist of DNA samples in solution inthe wells of a microtiter dish.

It also is contemplated by the inventor that one may “reverse” the abovedescribed detection protocols. For example, instead of using amplifiedinsertion junctions for preparation of a detectable array, one could usegenetic sequences which are specific to the locus for which an insertionmutation is desired. In this case, one could label the amplifiedinsertion junctions and use then as probes for the detection of locicorresponding to the insertion mutation. Therefore, by multiplehybridizations with different pools of amplified insertion junctions,one may ultimately identify individuals having the desired insertionmutations.

As an alternative to detection of insertion junctions with PCR orhybridizations, sequencing of insertion junctions may be used. In thisprocedure one would preferably first prepare pools of DNA fromindividuals having insertion junctions. The pools may be designed suchthe source of a particular insertion junction can be identified withoutthe need for screening of all individuals within a population. Anexemplary pooling procedure comprises the designation of individualsinto a 2×2 grid. Pools of DNA are then prepared from all of theindividuals within each column and row. The identification of a sequencein a column and a row will thereby provide a precise coordinate for theindividual having that sequence. Alternatively, pools needn't be used,however, this will be less preferred as more effort will be needed tofind a specific desired insertion.

III. Competitive Hybridizations

Use of the current invention may, in particular circumstances, requirecompetitive hybridizations. This may be so when the locus-specific probeused contains one or more sequences which are repeated throughout thetarget genome, thereby leading to detection of multiple, non-specificloci. The situation will arise more frequently where probes are derivedfrom genomic DNA clones of organisms which have relatively large genomessuch as many mammals, and particularly plants such as maize and wheat.

Signal from repetitive sequences may be “blocked” by inclusion ofunlabeled total genomic DNA in the mixture of labeled probe DNA, or byuse of the unlabeled DNA in prehybridizations before application of thelabeled probe. Even more effective than total genomic DNA for blockingwill be DNA which is “enriched” for repetitive, such as C_(o)t-1 DNA(Zwick et al., 1997, specifically incorporated herein by reference inits entirety). It is also contemplated that one may wish to use blockingDNA which contains unlabeled sequences of the insertional mutagen. Thismay help to avoid detection of the insertional mutagen and help ensureonly detection of the flanking sequences.

The proportion of blocking DNA to probe DNA used will vary and willdepend on a number of variables. Factors upon which the concentrationused is dependent include: the relative proportion of repetitivesequences in the probe/primer and target sequences, the desired level ofsensitivity in the detection, the size of the repetitive sequences, andthe degree of sequence homology between the probe repetitive sequencesand those of the target. Typical concentrations of unlabeled blockingDNA which may be used include from about 20 to about 200 fold excess,relative to the probe, including about 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, and 190 fold excess,Alternatively, one may wish to use concentrations of blocking DNAgreater or lesser than this range, including about 10, 300, 400, 500,600, 700, 800, 900, or about 1000 fold excess. The optimal concentrationused, however, will be dependent on the above mentioned factors and willbe known to those of skill in the art in light of the presentdisclosure. It is noted, however, that while competitive hybridizationsare effective in eliminating background signal caused by repetitivesequences, it will be preferable to avoid the problem through use ofunique or low copy probe sequences, such as, for example, cDNAs.

IV. Use of the Invention for Discovery of Gene Function

An important use of the current invention will be in acquiringinformation regarding the function of genes. Therefore, one embodimentof the invention involves the identification and isolation of a mutantfor a selected gene and the use of that mutant in studies of genefunction. By comparison of the phenotype of one or more individualshaving a particular insertion mutation to a representative sample ofindividual without the mutation, inferences may be made regarding thefunction of the mutated sequence.

In this manner, one may begin with a cDNA or other probe or primerspecific for a genetic sequence of unknown function, and, through use ofthe current invention, obtain information regarding the function of thatsequence. In light of the high-throughput-capability of the currentinvention, one could, alternatively, systematically obtain large numbersof mutants and screen the mutants for identification of genes associatedwith traits of interest. For example, one may use a sample of plant cDNAprobes to isolate maize plants having mutations corresponding the cDNAs.These mutants may then be grown in the field and various observationsmade of the mutant phenotype including characteristics such as yield,disease or pest resistance, stress tolerance, or any other trait deemedof interest. A correlation between a particular mutant and a phenotypewill, of course, suggest that the mutated gene is involved in theexpression of that trait. The mutated gene can then be cloned or usedfor further studies as desired by the user of the invention. Suchstudies may involve, for example, operably linking the cloned gene to adifferent promoter and using the construct created to transform plants.

V. Expression Analysis

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA will only be expressed in particular cells ortissue types, and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR techniques may also be used for detection andquantitation of RNA produced from introduced genes. In the applicationof PCR, it is first necessary to reverse transcribe RNA into DNA, usingenzymes such as reverse transcriptase, and then, through the use ofconventional PCR techniques amplify the DNA. In most instances, PCRtechniques, while useful, will not demonstrate the integrity of the RNAproduct. Further information about the nature of the RNA product may beobtained by Northern blotting. This technique will demonstrate thepresence of an RNA species and give information about the integrity ofthat RNA. The presence or absence of an RNA species can also bedetermined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and will onlydemonstrate the presence or absence of an RNA species.

While Southern blotting and PCR may be used to detect the gene(s) inquestion, they do not provide information as to whether the gene isbeing expressed. Expression may be evaluated by specifically identifyingthe protein products of the introduced genes or evaluating thephenotypic changes brought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques, such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity, such as western blotting, in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest, such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Assay procedures also may be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to be analyzedand may include assays for PAT enzymatic activity by followingproduction of radiolabeled acetylated phosphinothricin fromphosphinothricin and ¹⁴C-acetyl CoA or for anthranilate synthaseactivity by following loss of fluorescence of anthranilate, to name two.

Very frequently, the expression of a particular mutant is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms, including, but not limited, to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of genesencoding enzymes or storage proteins which change amino acid compositionand may be detected by amino acid analysis, or by enzymes which changestarch quantity, which may be analyzed by near infrared reflectancespectrometry.

VI. Genetic Characterization of Insertional Mutants

To confirm the presence of one or more insertional mutants in anindividual, to track these in progeny, and to analyze the effects of aparticular mutation, a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays, such as Southernand Northern blotting and PCR; “biochemical” assays, such as detectingthe presence or absence of a particular protein product, e.g., byimmunological means (ELISAs and Western blots) or by enzymatic function;plant part assays, such as leaf or root assays; and also by analyzingthe phenotype of the whole regenerated plant.

(i) DNA Integration, RNA Expression and Inheritance

Genomic DNA may be isolated from any plant or animal cells to determinethe presence of a particular insertional event using techniques wellknown to those skilled in the art. The presence of an insertional mutantmay, for example, be determined by polymerase chain reaction (PCR).Using this technique, discrete fragments of DNA are amplified anddetected by gel electrophoresis. This type of analysis will permit oneto follow a particular insertional mutant in the offspring of a cross.Insertional mutants are expected to be generated randomly and, for thisreason, are expected to be unique, based on their genomic location.Thus, by designing PCR primers which will amplify segments which includeboth the inserting DNA and the subsequently mutated native sequence,unique amplification products which are specific to that insertion eventcan be identified.

Southern hybridization is especially useful for identification ofparticular insertional mutants, in that each insertional mutant isexpected to have a unique restriction pattern. Using this techniquespecific, DNA sequences that were introduced into the host genome andflanking host DNA sequences can be identified. Hence, the Southernhybridization pattern of a given insertion event serves as anidentifying characteristic of that transformant. The technique ofSouthern hybridization provides information that is obtained using PCR,e.g., the presence of an integration event, but also characterizes eachindividual insertion event.

Both PCR and Southern hybridization techniques can be used todemonstrate transmission of an insertional mutant to progeny. In mostinstances, the characteristic Southern hybridization pattern for a giveninsertional mutation will segregate in progeny as one or more Mendeliangenes (Spencer et al., 1992), indicating stable inheritance of thetransgene.

For use as a probe, one may use DNA of the insertional mutagen, from themutated endogenous sequence, or from both. In the case of an insertionalmutagen which is present in low copy, it may be desirable to use DNAfrom the insertional mutagen as a probe. However, where the insertionalmutagen is present in high copy, such as will be the case withendogenous transposable elements, the detected restriction patterns willbe complex and difficult to interpret. In this case, it may be desirableto use the endogenous, mutated sequence as a probe.

The biological sample for assays may potentially be any type of plant oranimal tissue. Nucleic acid may be isolated from cells contained in thebiological sample, according to standard methodologies (Sambrook et al.,1989). The nucleic acid may be genomic DNA or fractionated or whole cellRNA. Where RNA is used, it may be desired to convert the RNA to acomplementary DNA. In one embodiment, the RNA is whole cell RNA; inanother, it is poly-A RNA. Normally, the nucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g.. ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of a radiolabel orfluorescent label, or even via a system using electrical or thermalimpulse signals (Affymax Technology; Bellus, 1994).

Following detection, one may compare the results seen in a given mutantwith a statistically significant reference group of non-mutatedcontrols. Typically, the non-mutated control will be of a geneticbackground similar to the mutated individual. In this way, it ispossible to detect differences in the amount or kind of protein detectedin various different mutants.

A variety of different assays are contemplated in the screening ofinsertional mutants isolated using the methods of the current invention.These techniques can be used to detect for both the presence ofparticular mutations as well as the resulting effects caused by themutations. The techniques include, but are not limited to, fluorescentin situ hybridization (FISH), direct DNA sequencing, PFGE analysis,Southern or Northern blotting, single-stranded conformation analysis(SSCA), RNAse protection assay, allele-specific oligonucleotide (ASO),dot blot analysis, denaturing gradient gel electrophoresis, RFLP, andPCR-SSCP.

(ii) Primers, Probes and Template-Dependent Amplifications

The term primer, as defined herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom 10 to 20 base pairs in length, but longer sequences can beemployed. Primers may be provided in double-stranded or single-strandedform, although the single-stranded form is preferred. Probes are defineddifferently, although they may act as primers. Probes, while perhapscapable of priming, are designed to bind to the target DNA or RNA andneed not be used in an amplification process. In preferred embodiments,the probes or primers are labeled with radioactive species (³²P, ¹⁴C,³⁵S, ³H, or other label), with a fluorophore (rhodamine, fluorescein),an antigen (biotin, streptavidin, digoxigenin), or a chemiluminescent(luciferase).

A number of template-dependent processes are available to amplify thesequences present in a given sample. One of the best known amplificationmethods is the polymerase chain reaction which is described in detail inU.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, each specificallyincorporated herein by reference in its entirety.

Briefly, in PCR, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the template to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR amplification procedure may be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described by Sambrook etal. (1989). Alternative methods for reverse transcription utilizethermostable, RNA-dependent DNA polymerases and are described in WO90/07641, filed Dec. 21, 1990.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in European Patent No. 0 320 308, specifically incorporatedherein by reference in its entirety. In LCR, two complementary probepairs are prepared, and, in the presence of the target sequence, eachpair will bind to opposite complementary strands of the target such thatthey abut. In the presence of a ligase, the two probe pairs will link toform a single unit. By temperature cycling, as in PCR, bound ligatedunits dissociate from the target and then serve as “target sequences”for ligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes amethod similar to LCR for binding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Patent Publication No. PCT/US87/00880,may also be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA that has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site, may also be useful in the amplification of nucleicacids in the present invention (Walker et al., 1992).

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR), involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3′ and 5′ sequences of non-specific DNA and a middlesequence of specific RNA is hybridized to DNA that is present in asample. Upon hybridization, the reaction is treated with RNase H, andthe products of the probe are identified as distinctive products thatare released after digestion. The original template is annealed toanother cycling probe, and the reaction is repeated.

Still another amplification method, described in GB Application No. 2202 328 and in PCT Patent Publication No. PCT/US89/01025 (eachspecifically incorporated herein by reference in its entirety), may beused in accordance with the present invention. In the formerapplication, “modified” primers are used in a PCR-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes is added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al.; PCTPatent Publication No. WO 88/10315; each specifically incorporatedherein by reference in its entirety). In NASBA, the nucleic acids can beprepared for amplification by standard phenol/chloroform extraction,heat denaturation of a clinical sample, treatment with lysis buffer andminispin columns for isolation of DNA and RNA or guanidinium chlorideextraction of RNA. These amplification techniques involve annealing aprimer which has target specific sequences. Following polymerization,DNA/RNA hybrids are digested with RNase H while double-stranded DNAmolecules are heat denatured again. In either case, the single-strandedDNA is made fully double-stranded by the addition of a second targetspecific primer, followed by polymerization. The double-stranded DNAmolecules are then multiply transcribed by an RNA polymerase, such as T7or SP6. In an isothermal cyclic reaction, the RNA's are reversetranscribed into single-stranded DNA, which is then converted todouble-stranded DNA, and then transcribed once again with an RNApolymerase, such as T7 or SP6. The resulting products, whether truncatedor complete, indicate target specific sequences.

European Patent Application No. 0 329 822 (specifically incorporatedherein by reference in its entirety) discloses a nucleic acidamplification process involving cyclically synthesizing single-strandedRNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be usedin accordance with the present invention. The ssRNA is a template for afirst primer oligonucleotide, which is elongated by reversetranscriptase (RNA-dependent DNA polymerase). The RNA is then removedfrom the resulting DNA:RNA duplex by the action of ribonuclease H (RNaseH, an RNase specific for RNA in duplex with either DNA or RNA). Theresultant ssDNA is a template for a second primer, which also includesthe sequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase 1), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle, leading to very swift amplification. With the properchoice of enzymes, this amplification can be done isothermally withoutthe addition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

PCT Patent Publication No. WO 89/06700 (specifically incorporated hereinby reference in its entirety) discloses a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” and “one-sidedPCR” (Frohman, 1990; Ohara et al., 1989; each specifically incorporatedherein by reference in its entirety).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide,” thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention (Wu etal., 1989, specifically incorporated herein by reference in itsentirety).

(iii) Detection Methods

Products may be visualized in order to confirm amplification of themarker sequences. One typical visualization method involves staining ofa gel with ethidium bromide and visualization under UV light.Alternatively, if the amplification products are integrally labeled withradio- or fluorometrically-labeled nucleotides, the amplificationproducts can then be exposed to X-ray film or visualized under theappropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by a labeled probe. The techniquesinvolved are well known to those of skill in the art and can be found inmany standard books on molecular protocols (see Sambrook et al., 1989).For example, chromophore or radiolabeled probes or primers identify thetarget during or following amplification.

One example of the foregoing is described in U.S. Pat. No. 5,279,721(specifically incorporated herein by reference in its entirety), whichdiscloses an apparatus and method for the automated electrophoresis andtransfer of nucleic acids. The apparatus permits electrophoresis andblotting without external manipulation of the gel and is ideally suitedto carrying out methods according to the present invention.

In addition, the amplification products described above may be subjectedto sequence analysis to identify specific kinds of variations usingstandard sequence analysis techniques. Within certain methods,exhaustive analysis of genes is carried out by sequence analysis usingprimer sets designed for optima sequencing (Pignon et al., 1994). Thepresent invention provides methods by which any or all of these types ofanalysis may be used.

(iv) Design and Theoretical Considerations for Relative QuantitativeRT-PCR.

Reverse transcription (RT) of RNA to cDNA followed by relativequantitative PCR (RT-PCR) can be used to determine the relativeconcentrations of specific mRNA species isolated from plants. Bydetermining that the concentration of a specific mRNA species varies, itis shown that the gene encoding the specific mRNA species isdifferentially expressed.

In PCR, the number of molecules of the amplified target DNA increases bya factor approaching two with every cycle of the reaction until somereagent becomes limiting. Thereafter, the rate of amplification becomesincreasingly diminished until there is no increase in the amplifiedtarget between cycles. If a graph is plotted in which the cycle numberis on the X axis and the log of the concentration of the amplifiedtarget DNA is on the Y axis, a curved line of characteristic shape isformed by connecting the plotted points. Beginning with the first cycle,the slope of the line is positive and constant. This is said to be thelinear portion of the curve. After a reagent becomes limiting, the slopeof the line begins to decrease and eventually becomes zero. At thispoint, the concentration of the amplified target DNA becomes asymptoticto some fixed value. This is said to be the plateau portion of thecurve.

The concentration of the target DNA in the linear portion of the PCRamplification is directly proportional to the starting concentration ofthe target before the reaction began. By determining the concentrationof the amplified products of the target DNA in PCR reactions that havecompleted the same number of cycles and are in their linear ranges, itis possible to determine the relative abundances of the specific mRNAfrom which the target sequence was derived can be determined for therespective tissues or cells. This direct proportionality between theconcentration of the PCR products and the relative mRNA abundances isonly true in the linear range of the PCR reaction.

The final concentration of the target DNA in the plateau portion of thecurve is determined by the availability of reagents in the reaction mixand is independent of the original concentration of target DNA.Therefore, the first condition that must be met before the relativeabundances of a mRNA species can be determined by RT-PCR for acollection of RNA populations is that the concentrations of theamplified PCR products must be sampled when the PCR reactions are in thelinear portion of their curves.

The second condition that must be met for an RT-PCR experiment tosuccessfully determine the relative abundances of a particular mRNAspecies is that relative concentrations of the amplifiable cDNAs must benormalized to some independent standard. The goal of an RT-PCRexperiment is to determine the abundance of a particular mRNA speciesrelative to the average abundance of all mRNA species in the sample.

Most protocols for competitive PCR utilize internal PCR standards thatare approximately as abundant as the target. These strategies areeffective if the products of the PCR amplifications are sampled duringtheir linear phases. If the products are sampled when the reactions areapproaching the plateau phase, then the less abundant product becomesrelatively over-represented. Comparisons of relative abundances made formany different RNA samples, such as is the case when examining RNAsamples for differential expression, become distorted in such a way asto make differences in relative abundances of RNAs appear less than theyactually are. This is not a significant problem if the internal standardis much more abundant than the target. If the internal standard is moreabundant than the target, then direct linear comparisons can be madebetween RNA samples.

The above discussion describes theoretical considerations for an RT-PCRassay for plant tissue. The problem inherent in plant tissue samples arethat they are of variable quantity (making normalization problematic),and that they are of variable quality (necessitating theco-amplification of a reliable internal control, preferably of largersize than the target). Both of these problems are overcome if the RT-PCRis performed as a relative quantitative RT-PCR with an internal standardin which the internal standard is an amplifiable cDNA fragment that islarger than the target cDNA fragment and in which the abundance of themRNA encoding the internal standard is roughly 5 to 100-fold higher thanthe mRNA encoding the target. This assay measures relative abundance,not absolute abundance of the respective mRNA species.

Other studies may be performed using a more conventional relativequantitative RT-PCR assay with an external standard protocol. Theseassays sample the PCR products in the linear portion of theiramplification curves. The number of PCR cycles that are optimal forsampling must be empirically determined for each target cDNA fragment.In addition, the reverse transcriptase products of each RNA populationisolated from the various tissue samples must be carefully normalizedfor equal concentrations of amplifiable cDNAs. This consideration isvery important since the assay measures absolute mRNA abundance.Absolute mRNA abundance can be used as a measure of differential geneexpression only in normalized samples. While empirical determination ofthe linear range of the amplification curve and normalization of cDNApreparations are tedious and time consuming processes, the resultingRT-PCR assays can be superior to those derived from the relativequantitative RT-PCR assay with an internal standard.

One reason for this advantage is that, without the internalstandard/competitor, all of the reagents can be converted into a singlePCR product in the linear range of the amplification curve, thusincreasing the sensitivity of the assay. Another reason is that, withonly one PCR product, display of the product on an electrophoretic gelor another display method becomes less complex, has less background, andis easier to interpret.

(v) Chip Technologies

Specifically contemplated by the present inventor are chip-based DNAtechnologies such as those described by Hacia et al. (1996) andShoemaker et al. (1996). Briefly, these techniques involve quantitativemethods for analyzing large numbers of genes rapidly and accurately. Bytagging genes with oligonucleotides or using fixed probe arrays, one canemploy chip technology to segregate target molecules as high densityarrays and screen these molecules on the basis of hybridization (seealso, Pease et al., 1994; and Fodor et al., 1989).

VII. Definitions

Corresponding Individual Lacking an Insertion Mutation: an individualwhich has the same genetic background as another individual, but differson the basis of lacking a particular insertion mutation.

Detectable Array: an arrangement of nucleic acid sequences from whichspecific sequences or subsets of sequences can be identified. The arraycan comprise DNA sequences bound to a solid support and can also includeDNA compositions arranged in solution in suitable containers. For thepurposes of the current invention the sequences will be ones which maybe used to identify one or more specific insertion junctions. Thesesequences can, therefore, represent DNA of insertion junctions or,alternatively, sequences representing a particular locus for which aninsertion mutation is desired.

DNA Composition Enhanced for a Plurality of Insertion Junctions: a DNAcomposition in which a non-locus specific selection of insertionjunctions has been enhanced relative to the starting DNA from which theDNA composition is derived. Such non-locus specific selections areprepared without the need for use of probes or primers which arespecific to the locus or loci for which an insertion mutation isdesired. The selection procedure will typically, instead, use probes orprimers which are specific to the insertional mutagen. Examples of suchprocedures include inverse PCR, primer adapted PCR, and vectorette PCR,AIMS, or any other amplification or isolation procedure which is capableof being used to enhance a DNA composition for a diverse class ofinsertion junctions.

Hybridization Filter: an object to which nucleic acids can be fixedlyattached, and to which probes may be hybridized, for example, inSouthern Hybridization. Exemplary hybridization filters will be made ofnitrocellulose or nylon, although any similar materials may also beused.

Insertion Junction: the segment of DNA encompassing the end of aninsertional mutagen and particularly, the flanking genomic DNA into theinsertional mutagen has inserted. For the purposes of the invention, DNAfrom the insertional mutagen itself need not typically be present, butfor detection, the flanking genomic DNA should be.

Insertional Mutagen: any sequence which is capable of inserting into asegment of genomic DNA thereby causing an insertion mutation.

Microscope Slide: an object similar to a standard slide used for holdinga specimen to be observed under a microscope. The microscope slide willpreferably be made of glass or a similar material and will have a flatsurface, however, it will be understood to those of skill in the artthat various trivial modifications may be made to a typical microscopeslide and still not depart from the scope and meaning of the term asdefined in the current invention.

Pool: a composition of DNA made from the combination of DNA frommultiple individuals. The pool will typically be constructed to allowthe identification of individuals possessing a desired genetic sequencefrom a populations of individuals without necessitating screening ofevery individual within that population.

VIII. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

EXAMPLE 1 Considerations in the Preparation of Arabidopsis InsertionalMutation Populations

A project was initiated to saturate the Arabidopsis genome withinsertion mutations. Based on the Arabidopsis genome size (100 Mb) andan average gene target size (2 kb), it was calculated that 100,000random insertions would make hitting any unique gene segment (2 kb) aprobable event (p>90%), assuming integration sites are chosen randomly.

Individual Arabidopsis plants were vacuum infiltrated according toBechtold et al. 1993, allowed to set seed, and seed was plated todetermine the frequency and pattern of transformation events.Independent insertions were selected with application of FINALE®(glufosinate herbicide). The transformation frequency, based on thetotal number of seed, was between 1 and 2%. Examination of severalhundred individual siliques indicated that, based upon T-DNAhybridization patterns, most, if not all, transformed plants werederived from independent T-DNA transformation events.

The T₁ transgenic plants contained between 1 and 20 T-DNA hybridizingbands. Some of these bands represent junction fragments between tandem(direct and inverted) repeats of T-DNA, while others represented uniquejunction fragments between plant DNA and T-DNA. Several plants wereoutcrossed to wild-type plants, and the T₂ outcross progeny wereexamined by Southern analysis to determine the number of independentloci based on recombination between T-DNA bands. By examining largenumbers of progeny crosses, it was ascertained that most T₁ plantscontained between 1 and 5 independent T-DNA loci.

It was thus shown that: 1) transgenic Arabidopsis can be directlyselected in soil using phosphinothricin resistance; 2) the frequency oftransformation averaged 1.5% of the total seed; 3) that most, if notall, T₁ resistant plants represented independent transformation events;and 4) that T₁ plants contained an average of 3 independent T-DNAinsertion loci per genome Therefore, to generate 100,000 independentinsertions, about 35,000 phosphinothricin-resistant (i.e., transformed)T₁ plants are needed. At an average transformation frequency of 1.5% anda total seed population of about 5,000 seeds per plant, it was decidedto vacuum infiltrate about 2,000 To plants to achieve saturation.

EXAMPLE 2 Generation of Arabidopsis Insertional Mutants and DNA Pools

Five to six Arabidopsis thaliana seeds were germinated in pots and grownat 21° C. under 16 hr light. Primary bolts were removed, and whensecondary bolts emerged, the plants were vacuum infiltrated with anAgrobacterium strain harboring the T-DNA containing the bar gene drivenby the constitutive viral promoter CaMV35S (Bechtold et al., 1993; Whiteet al. 1990; SEQ ID NO:1). A total of over 2,000 plants were vacuuminfiltrated and allowed to self-pollinate. Seeds were collected fromindividual pots, vernalized, and germinated in soil at a density ofapproximately 10,000 seeds per pot. After seedlings emerged, they weresprayed with FINALE® herbicide (BASF Inc.) 1 time per week, for up to 6weeks, using the manufacturers recommended level of application.Non-transformed plants died, while transgenic plants thrived underselection.

When the primary bolts emerged from the 100-150 resistant T₁ plants ineach pot, tissue was harvested for DNA extraction to generate the T₁ DNApool. For DNA extraction, four to sixteen leaf punches were placed ineach tube of a 96 cluster tube rack (CoStar Inc., Cat#4410). Sampleswere cooled in a liquid nitrogen bath, or alternatively, lyophilizedovernight and ground to a powder with a wooden stick or glass rod on dryice. Following grinding, 5 zirconium beads were added (2.5 mm, ZirconiaSilica Beads, Biospec Products, Inc.) to each tube and the samplescapped (Microplate Sealers Titer Tops from Diversified Biotech, Catalog#TTOPS). The sample plate was placed onto a bead beater (BiospecProducts, Inc.) and shaken for 1 min on medium setting. Then 0.5 ml ofprewarmed (65° C.) extraction buffer (100 mM Tris pH8, 50 mM EDTA, 1%SDS, 500 mM NaCl) was added, the samples capped, vortexed, and allowedto incubate at 65° C. for 10 min in a water bath. One hundred sixtymilliliters cold 5 M Kac was added, and samples were capped, mixed byinverting, and placed on ice for 5 min. The tube rack was then spun at3000 rpm for 10 min in a table top plate carrier.

The supernatant (300 ml) was then transferred to a filtaplate (course,96 well 300 μl FiltaPlate Plus; Polyfiltronics FP350PSC/CF/D) and 300 ml4.4M NH₄OAc/Isopropanol (1:7 ratio) was added with a 20 ml Quick-PrecipPlus (AGTC 72641) to an 800 ml receiver plate (96 well 800 μl Receiverplates, AGTC 22304). The filter plate was stacked on the receiver plateand the crude extract spun into a capture plate at 3000 rpm for 5 min.The capture plate was capped and mixed by an inverting spin at 3000 rpm10 min. The plate was inverted to empty isopropanol, 200 μl 70% EtOH wasadded to pellet the DNA, and the plate was inverted to empty the EtOH,followed by air drying of the pellet. Once the pellet was dry, 100 ml ofTE (TE (10/1) pH 8+0.4 mg/ml Rnase) is added, the samples covered andvortex on slow. The DNA is stored at 4° C.

The T₁ plants were then allowed to mature and set seed to generatecorresponding T₂ seed pools. A total of 384 pools of 100-150 T₁ plantswere produced along with T₁ DNA and T₂ seed pools. Each pool representedan average of 125 plants containing, on average, 4 independentinsertions per genome, or a estimated total of over 203,650 T-DNAinsertions.

The population of Arabidopsis thaliana plants having insertion mutationswas organized in Collections, Sets, and Pools of T₁ DNA and T₂ seed.Collections (a, b, c, . . . ) were defined by the T-DNA construct (i.e.,Collection “a” contains the 35S::bar gene and a synthetic supF gene forjunction fragment rescue; SEQ ID NO:1). Each Collection consisted ofthree or more Sets (1, 2, 3, . . . ) of 96 Pools (designatedalphanumerically A01-H12) per Set. Hence, Collection “a” consists of 384pools labeled a1.A01 through a3.H12. Each pool, represents approximately300-500 independent T-DNA insertions. Hence, a Collection contains atotal of 288 Pools (285 transgenic pools plus 3 control pools) andrepresents approximately 85,000 to 140,000 independent T-DNA insertions.

EXAMPLE 3 Confirmation of the Generated Population of ArabidopsisInsertional Mutants

To confirm that the generated population contained the predicted numberof insertional mutants, standard site-selected mutagenesis was appliedto locate insertions in several genes of interest, including the twocytosine DNA methyltransferase genes (MET1 and MET2). First, Set a1(pools a1.A01 through a1.H12) was screened using a PCR reactioncontaining a gene-specific primer designed to the 3′ UTR together with aright border T-DNA primer designed just inside the right borderjunction.

The Set a1 T₁ DNA pools were screened with one of the four gene-specificprimers together with the right border primer. Five microliters of thePCR reactions was denatured and applied to a membrane in a 96-wellmanifold, and membranes were hybridized with the appropriategene-specific probes. In each case, from 1 to 3 insertion alleles couldbe detected for all four genes from Set a1 pools, a result consistentwith the estimate of T-DNA insertion copy number. If the left borderprimer detects a similar number of insertions in opposite orientationwith respect to transcription, it is estimated that the YATDL collectionwould contain between 8 and 24 alleles for each Arabidopsis gene. Thisassumes all genes are targets for T-DNA insertion. If the four chosentargets were typical ones, any two Sets (i.e., a1 and a2) should containat least two alleles in most Arabidopsis genes.

EXAMPLE 4 Enriching for Mu-Tagged Sites by Amplification of InsertionMutagenized Sites (AIMS)

Maize plants having Mu insertion mutations are organized into 32×32grids. DNA is then extracted from individual maize plants using theprocedure of Example 19, and pools of the DNA are made for each row andcolumn. The pooled DNA is digested either with the restrictionendonuclease Bfal or the enzyme Msel. For restriction of 500 ng DNA, 5 Uof Msel or Bfal is placed in a 40 μl volume of 1×RL-Buffer for 1 h at37° C. (1×RL contains 10 mM Tris-Acetate, pH 7.5, 10 mM Mg-Acetate, 50mM K-Acetate, and 50 ng/μl BSA). Linker sequences (Msel/Bfal) areligated by adding together 1 μl 50 μM Msel or Bfal adapter, 1 μl10×Ligation Buffer (Boehringer), 1 U T4-DNA Ligase, and water to a finalvolume of 50 μl, followed by incubation for at least 2 h at 37° C.(European Patent application 92402629, specifically incorporated hereinby reference in its entirety). For amplification of the Mu-elementinsertion sequences, a linear PCR is performed using a biotinylatedprimer complementary to the Mu-element ends (Mu-Bio), and theamplification product is separated with streptavidin-coated magneticbeads. The PCR mix for the linear amplification is composed ofapproximately 27.5 μl DNA, 2.5 μl 12 μM Mu-Biotin primer, 10 μl 2.5 mMdNTPs (each), 5 μl 10×KCl V buffer, and 1 U Taq DNA polymerase(Boehringer) in a final volume of 50 μl. Amplification is carried out in12 cycles using the following PCR program:

1: 94° C. 3 min 2: 94° C. 1 min 3: 65° C. 30 sec 4: 72° C. 60 sec

cycle 4 to 2, 11 times (Use of more than 12 cycles can cause aberrantexponential PCR amplification)

6: 72° C. 3 min.

Primer and adapter sequences (‘-3’ orientation) are as follows:

Mser/Bfal Adapter:

TACTCAGGACTCAT (SEQ ID NO:2)

GACGATGAGTCCTGAG (SEQ ID NO:3)

Mu-Bio: AGAGAAGCCAACGCCA(A/T)CGCCTCCATT (SEQ ID NO:4)

Msel Sel/A(GCT): GATGAGTCCTGAGTAA/A(GCT) (SEQ ID NO:5)

Bfal Sel/A(GCT): GATGAGTCCTGAGTAG/A(GCT) (SEQ ID NO:6)

Mu Sel: TCTATAATGGCAATTATCTC (SEQ ID NO:7)

For removal of excess Mu-Biotin primer, a QIA-quickspin column is usedas follows: add 250 μl PB buffer to 50 μl PCR reaction; spin; washcolumn with 2600 μl PE; elute with 50 μl TE, pH 8.5; add 50 μl 4 M NaClto 50 μl eluat; spin down briefly; use directly for PCR. The isolatedbiotin-labeled sequences are amplified by PCR with Bfal or Msellinker-specific primer (Msel Sel/A or Bfal Sel/A). The radioactivelabeled nested Mu-specific primer (Mu Sel) is prepared in 20 reactions,each containing 2.5 μl of 10 μM Mu Sel, 5 μl Gamma ATP (50 μCi), 1.25 μlOne Phor All+buffer (Pharmacia), and 1-2.5 U T4 Polynucleotide Kinase,in a total volume of 12.5 μl. To lower the complexity of the amplifiedsequences, the linker-specific primer has a one nucleotide extension atits 3′-end, and individual reactions are made for all eight linkerprimers. Exponential PCR is carried out with the labeled primer in areaction containing 5 μl beads/DNA (suspend well before pipetting), 0.5μl labeled Mu Sel Primer, 0.6 μl 10 μM Msel Sel/N or Bfal Sel/N, 4.0 μl2.5 mM dNTPs, 2.0 μl 10×Ammonium sulphate buffer, and 2.0 μl BSA 1 mg/mlin a final volume of 17 μl, covered with paraffin. The PCR program is asfollows:

1) 94° C. pause add 1 U Taq polymerase in 3 μl volume and continue 2)94° C. 1 min 3) 65° C. 30 sec decrease by 0.7° C. every cycle 4) 72° C.1 min cycle 4 to 2 18x 5) 94° C. 1 min 6) 52° C. 30 sec 7) 72° C. 1 mincycle 7 to 5 26x 8) 72° C. 3 min

Following amplification of the Mu insertion junctions, the DNA may beused for preparation of arrays and subsequent detection of insertionalmutants. Alternatively, Mu-tagged sites may be amplified usingvectorette PCR, IPCR, or other techniques.

EXAMPLE 5 Amplification of Insertion Junctions Using Primer-adapted PCR

Sau3AI-digested genomic DNA (50 μg) is separated on a 1% agarose gel,and size fractions of approximately 500 and 900 bp are excised from thegel and purified using a GeneClean® kit (Bio 101, La Jolla, Calif.).Ligation of adapters is performed in a total volume of 20 μl ofadapter-ligation buffer (66 mM Tris-HCl, pH 7.6; 10 mM MgCl₂; 10 mMdithiothreitol [DTT]; 0.3 mM ATP; 1 mM spermidine-HCl; and 200 μg/mlbovine serum albumin [BSA]) with 200 ng adapters, 2-5 μg genomic DNA,and 1 U T4-DNA ligase (GIBCO BRL/Life Technologies, Gaithersburg, Md.).The ligation reaction is incubated at 16° C. overnight, and thenon-ligated adapters are removed by spin-column purification (Sephacryl®S-300, Pharmacia LKB Biotechnology AB, Uppsala, Sweden). The columns areequilibrated with PCR buffer (50 mM KCl; 10 mM Tris-HCl, pH 8.3; and 1.5mM MgCl₂) and eluted in 50 μl volume.

An insertional mutagen complementary primer is first used for a linearamplification of the insertion junction sequences. The reaction mixtureof 100 μl contains 200 μM deoxynucleoside triphosphates (dNTP), 1×PCRbuffer (Promega, Madison, Wis.), 20 pmol biotinylated primer 1, 30 μl ofadapter-ligated genomic DNA template, and 1 U Taq DNA polymerase(Promega). The temperature program is a two-step program of 50 cyclescomprising 95.5° C. for 30 s and 70° C. for 2 min 30 s on the PCRmachine PHC-2 (Techne, Cambridge, UK). The linear biotinylated productsare bound to Dynabeads® M-280 Streptavidin (Dynal A. S., Oslo, Norway);0.25 mg beads are washed prior to binding (twice with 1 M NaCl in TEbuffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) and once with 1×PCRbuffer) and then incubated for 5 min at room temperature (RT) with theamplified product. After binding, the supernatant containing the genomicDNA is removed by fixing the beads with the magnet MPC-E (Dynal) anddiscarding the supernatant. The beads are washed 3 times with 1 M NaClin TE buffer, 3 times with TE buffer, and once with PCR buffer.

Exponential PCR is then performed on the single-stranded template boundto the beads, with the 100 μl of reaction mixture containing 200 μMdNTP, 1×PCR buffer, 50 pmol of each of the adapter-primer and thenon-biotinylated primer 1, and 1 U Taq DNA polymerase. The temperatureprogram is 35 cycles of 95.5° C. for 30 s, 62° C. for 1 min, and 72° C.for 1 min. A second exponential PCR of 35 cycles is performed under thesame conditions as above using 1 μl of the obtained PCR product in 100μl reaction mixture and replacing primer 1 with the nested primer 2.

The PCR products may then be directly used for the preparation of arraysor can be “blunted” using Klenow polymerase (David et al., 1986) andsubcloned into the SmaI site of pBluescript® (Stratagene, La Jolla,Calif.). The inserts can then be completely sequenced on both strands orcan be used to transform bacteria for the production of additionalinsertion DNA.

EXAMPLE 6 Amplification of Insertion Junctions Using Vectorette PCR

Prepared DNAs are digested with appropriate restriction enzymes insuitable buffers at 37° C. for 1 h ATP, dithiothreitol (DTT) is added toa concentration of 2 mM, and appropriate vectorette (commerciallyavailable from Clonetech Inc., Palo Alto, Calif.) units are added alongwith 1 U T4-DNA ligase (without a change of buffer). The samples arethen incubated at 20° C. for 1 h followed by 37° C. for 30 min. Thisincubation cycle is carried out a further two times because thevectorettes are designed so that the restriction enzyme site is notreformed on ligation of the vectorette to target DNA; this incubationcycle leads to increased target-vectorette constructs. The incubation at37° C. leads to digestion of target-target DNA but not target-vectoretteconstructs. PCR is performed using the appropriate known biotinylatedprimer and vectorette PCR primer in 1×Taq PCR buffer with 2.5 U Taq DNApolymerase (Promega). PCRs may be carried out, for example, using aTechne PHG 1 unit, with 40 cycles of 96° C. for 1 min, 64° C. for 1 min,and 74° C. for 1.5 min. PCR products are visualized on a 1% agarose gelstained with ethidium bromide and/or used directly for arraypreparation.

EXAMPLE 7 Amplification of Insertion Junctions Using Inverse-PCR

Restriction digests are carried out using 5 μg of source DNA treatedwith 10 U EcoRI according to the supplier's specifications (U.S.Biochemicals). Digested DNAs are electrophoresed thorough a 1.1% (w/v)agarose gel (SeaKem) in 1×TBE buffer (50 mM Tris; 100 mM Borate; and 10mM EDTA, pH 8.2). Appropriate fragments are excised from the gel,electroeluted in 0.5×TBE, and extracted twice with phenol and once withchloroform; and the DNA concentration is determined by UVspectrophotometry.

For circularization, 0.1 μg of the appropriate restriction fragment isdiluted to a concentration of 0.5 μg/ml in ligation buffer (50 mM TrisHCl, pH 7.4; 10 mM MgCl₂; 10 mM dithiothreitol; 1 mM adenosinetriphosphate; and 10 μg/ml gelatin). This ligation reaction is initiatedby the addition of T4-DNA ligase (New England Biolabs) to aconcentration of 1 U/μl, and the reaction is allowed to proceed for 16 hat 12° C. The ligated sample is then treated with an equal volume ofphenol:chloroform mixture, the aqueous phase is removed, and the DNA isprecipitated with ethanol and collected by centrifugation.

The PCR is performed manually in reactions containing 0.1 μg ofcircularized DNA obtained as described above in the presence of 50 pmolof each primer and 500 μM dNTPs. The primers are synthesized using anApplied Biosystems automated oligonucleotide synthesizer. Thirty cyclesof denaturation are carried out at 94° C. for 1.5 min, followed byprimer annealing at 48° C. for 1.0 min and extension by Taq DNApolymerase (Perkin-Elmer Cetus) at 70° C. for 4.0 min. The resultingsample is desalted, and excess dNPTs are removed with a Centricon 30microconcentration column from Amicon (Higuchi et al., 1988; Saiki etal., 1988). The DNA products from the PCR reactions are then used forthe production of arrays for detection of specific insertional mutantsor cloned into appropriate vectors.

EXAMPLE 8 Screening T₁ DNA Pools by PCR

PCR screening may be used as an alternative to hybridization of griddedarrays of PCR-amplified T-DNA::plant junction fragments representinginsertions in all 384 T₁ DNA pools. Initially, two T₁ DNA Sets arescreened per gene (192 reactions) in a 192-well plate. This format ispreferred to a 384 format because each gene screen can be barcoded andhandled separately. This format can be easily managed with two roboticsunits, a Hydra 96 unit (Robbins Scientific, Sunnyvale Calif.) toefficiently dispense T₁ DNA templates and a Beckman Biomek 2000automatic liquid handling unit fitted with a chilled base to assemblePCR reactions. This strategy should, on average, identify between 2 to 6insertions per gene. Using two four-block PCR instruments (MJ ResearchPTC225), PCR screens may be performed on 8 genes per cycle, or a minimumof 40 genes per week.

Approximately 5 μl of the reaction is then denatured and applied to afilter membrane via a manifold apparatus. Gene-specific primers are usedto simultaneously amplify and radiolabel an appropriate region of theprovided cDNA clone using a PCR instrument dedicated for radioactivereactions. Primers are removed by spun columns, and the probe isdenatured and hybridized to the filter membranes overnight according toExample 11. The following day, the filters are washed and imaged using aphosphoimager.

EXAMPLE 9 Multiplexing T₂ Seed Pools

This is dependent upon whether or not a pool has previously beenmultiplexed. If it has, the T₂ screen starts directly with the PCR stepdetailed below. For non-multiplexed pools, providing T₃ seed harboringan insertion allele requires one additional Arabidopsis generation (8weeks from start to finish). The frequency of this delay decreases inproportion to the number of pools multiplexed, however. It will bepreferable to multiplex all pools and utilize direct T₂ screening tolocate the T₃ seed pool.

The number of T₂ plants needed to screen to have a 95% probability ofrecovering any particular insertion can be estimated. Assuming that thehomozygous condition is non-lethal, 480 T₂ plants are needed; should thehomozygous condition result in lethality, this number increases to 864plants.

Based on the calculations, the technique to multiplex T₂ pools is asfollows: approximately 1000 T₂ seeds are vernalized and suspended in0.1% agarose. The seed-containing solution is pipetted onto the surfaceof 96 pots to yield approximately 8-10 seeds per pot. After germination,seedlings are thinned back to 5 plants per pot, for a total of 480plants. This number gives a 95.8% chance of recovering a non-lethalallele and an 80.4% probability for recovering recessive lethals.Initially, this strategy is more preferred than screening two sets of480 (960) plants from a single T₁ pool because more time and resourcesare used generating independent T₂ DNA and T₃ seed pools. This meansthat more pools are available sooner and insertions may be more rapidlyidentified. Since 4-8 hits are expected per gene in a primary screen, ahigh probability of recovering missed lethals can be achieved by simplyscreening a different T₂ pool of 480 plants, rather than multiple setsfrom one T₂ pool.

At the time of bolting, tissue (one leaf per plant, 5 leaves per T₂pool) is placed into a deep 96-well plate and lyophilized. All T₂ DNAsamples are extracted simultaneously in a deep 96-well plate using thetechnique in Example 2, to yield enough high quality DNA for over 500PCR reactions. After seed set, the T₃ seed is collected from individualpots and stored in alphanumeric coordinates of a deep 96-well plate.

To identify the T₃ seed pool containing the insertion of interest, theT₂ DNA is pooled by row (8 individuals) and column (12 individuals), anda PCR screen (8+12=20 reactions per pool, 2 pools per gene for a totalof 40 PCR reactions per gene) and a dot blot hybridization are performed(alternatively, this step may be accomplished by gridding andhybridization to PCR-amplified junction fragments in a 96-array format).The row and column coordinate of the T₃ seed pool containing theinsertion allele of interest is determined by the hybridization pattern.Initially, two multiplex pools are chosen per gene to provide twoindependent alleles; more are done if needed (i.e., the insertion is notfound in the 480-plant multiplex pool).

EXAMPLE 10 High Density Filter Construction

Fifty to 100 ng of DNA from the pools of amplified insertion junctionsare placed in 96-well microtiter plates and dotted using the “Saturnin”robot of Genethon onto nylon filters of 8×12 cm (Hybond N+, AmershamCorporation, Amersham, UK) at an array density of 16 microtiter platesarrayed in a 4×4 format. DNA is cross-linked to the membrane byultraviolet radiation (120 mJ/cm²) using the Stratagene UV-Stratalinker2400 (Stratagene, LaJolla, Calif.). Control clones are also spotted atspecific positions on the filter. Membranes are prepared in batches andstored at 4° C. before use. The procedure is repeated until the desirednumber of sectors have been prepared.

EXAMPLE 11 Probe Preparation, Labeling, and Hybridization

The locus specific probe, comprising a single or low copy sequence islabeled with 50 μCi of [γ-³³]ATP (Amersham) (3000 Ci/mmole) using 10 UT4 polynucleotide kinase (Boehringer Mannheim, Mannheim, Germany) for 30min at 37° C. Filters are incubated within glass tubes in anhybridization oven (Appligene, Strasbourg, France) in a volume of 15 ml.Membranes in duplicate are prehybridized for 5 hr at 42° C. in a 15-mlsolution containing final concentrations of 4×SSC (1×SSC=150 mM NaCl and15 mM sodium citrate), 50% formamide, 10×Denhart's, 0.1% sodium dodecylsulfate (SDS), 8% dextran sulfate, 50 mM phosphate buffer (pH 7.2), and1 mM EDTA. Hybridization of the replicate set of filters is performedovernight at 42° C. in the same solution with 15 to 20×10⁶ cpm of³³P-radiolabeled probes in the presence of 100 μg/ml of denaturedherring sperm DNA. In the case of probes which contain one or morerepetitive sequences which may cause non-gene specific hybridization,some or all of the herring sperm DNA is replaced with either totalgenomic DNA or C₀t-1 DNA. This DNA will hybridize competitively with therepeated elements and effectively block their signal. The membranes arewashed twice for 10 min in 2×SSC/0.1% SDS, followed by washing once for15 min in 1×SSC/0.1% SDS and twice for 15 min in 0.1×SSC 0.1% SDS. Allwashes are carried out at 65° C. Exposure to phosphor screens is for 1to 3 days.

Stripping of hybridized membranes is performed by two successiveimmersions in a solution of 0.4 M NaOH and 0.1% SDS at 65° C. for 30min. Membranes are rinsed in 0.2 M Tris-HCl (pH 8.0) and 1×SSC/0.1% SDSfor 10 min at room temperature. Membranes may be used a minimum of 5 oftimes.

Hybridization of the membranes with the ³³P-radiolabeled oligonucleotideprobe is performed in 7% SDS, 0.5 M phosphate buffer (pH 7.2), and 1 mMEDTA for 15 hr at 50° C., followed by washing in 2×SSC for 15 min at 50°C., followed by 15 min at room temperature and a final wash in1×SSC/0.1% SDS for 15 min at 37° C.

EXAMPLE 12 Hybridization Signal Analysis

Filters are scanned on the Phosphorlmager imaging Plate system(Molecular Dynamics, Sunnyvale, Calif.) for quantitative analysis ofsignal intensities. After image acquisition, the scanned 16-bit imagesare imported on a Sun workstation and image analysis is performed usingthe XdotsReader software (Cose, Le Bourget, France).

The software processes the results of an exposure into images ofindividual filters and then translates the hybridization signalcoordinates into dot localization on the filter using a reference gridfor the arrangement of the dots. It takes into account slight variationsin dot position attributable to filter deformation by assigning thesignal detected to the nearest position expected. The softwarequantifies each dot individually after local background subtraction.These tasks, image cutting, dot identification, and dot quantificationare processed sequentially and automatically. The results are validatedinteractively, and a table is generated that contains for each dot itsreference number and the experimental values.

Different types of values may be obtained for the quantification of thedot intensity: the radius of the dot, the mean of the dot pixelintensities for one dot, the maximal intensity of the pixels of the dot,the sum of the pixel intensities of the dot, and the average of thepixel intensities of the dot weighted by the distance to the center ofthe dot. To take into account experimental variations in specificactivity of the probe preparations or exposure time that might alter thesignal intensity, the data obtained from different hybridizations may benormalized by dividing the Im for each dot by the average of theintensities of all the dots present of the filter to get a normalized Imvalue (nIm).

EXAMPLE 13 Rehybridization of Nucleic Acids toOligonucleotide-Derivatized Substrate Surface

The arrays are hybridized with labeled probe in accordance with theprocedure of Example 14, and then washed three times with a 100° C.solution of 0.01% SDS. The slides are then allowed to expose XAR filmovernight at −70° C. to confirm the labeled probe is removed.Rehybridization with the labeled probe is carried out using theStratagene Qwik-Hyb(tm) hybridization accelerator solution following thepackage insert directions. Other hybridization acceleration reagentsthat can be employed in the methods of the present invention include A1protein, RecA protein, SSB, dextran sulfate, ficoll, phenol, anddetergent.

Prehybridization is carried out for 15 minutes at 53.5° C., and then 100μl of 10 mg/ml salmon sperm DNA is added to the slides together with 10μl of the labeled probe. The hybridization reaction is carried out at53.5° C. for one hour. The slides are washed and then allowed to exposeXAR film overnight at −70° C.

EXAMPLE 14 Identifying Individual Arabidopsis Plants with InsertionAlleles

Once an appropriate T₃ seed pool has been identified, the last step isto find the individual plant(s) harboring the insertion. Southernanalysis of approximately 25 T₃ plants (p=97% chance of identifying ahomozygote or heterozygote in the T₃) is a preferred method for thispurpose.

Some types of mutations may not be found or may be difficult to find.For instance, dominant lethality or sterility mutations will be lost inthe T₁ generation and not present in the collection. Recessive lethalityor sterility is less problematic. There may be instances in which amutation is identified in the T₂ multiplex screen, but absent in the T₃seed—e.g. male or female sporophytic sterility will be undetected at thetime of tissue sampling. This problem may be avoided, however, becauseof the high number of expected hits (from 4-8). Since the phosphoimagerdata will be saved electronically, should no T₃ plant be found harboringthe mutation of interest, additional T₃ seed may simply be screened, oradditional T₂ DNA PCR screens may be used to identify additional T₃pools.

EXAMPLE 15 Introgression of an Insertion Mutation into Elite Inbreds andHybrids

It is specifically contemplated by the inventor that an insertionalmutation identified by the current invention may provide a plant with adesired characteristic and that one may therefore wish to move theinsertion mutation from one genetic background into another.Backcrossing may be used to achieve this goal. Backcrossing can be usedto transfer a specific trait from one source to an inbred that lacksthat trait. This can be accomplished, for example, by first crossing asuperior inbred (A) (recurrent parent) to a donor inbred (non-recurrentparent), which carries the appropriate mutation. The progeny of thiscross are first selected in the resultant progeny for the mutation to betransferred from the non-recurrent parent, and then the selected progenyare mated back to the superior recurrent parent (A). After five or morebackcross generations with selection for the desired trait, the progenyare hemizygous for the mutant loci, but are like the superior parent formost or almost all other genes. The last backcross generation would beselfed to give progeny which are pure breeding for the insertionalmutagen(s) being transferred, i.e., one or more integration events.

EXAMPLE 16 Marker-Assisted Selection

Genetic markers may be used to assist in the introgression of one ormore integration events from one genetic background into another.Marker-assisted selection offers advantages relative to conventionalbreeding in that it can be used to avoid errors caused by phenotypicvariations. Further, genetic markers may provide data regarding therelative degree of elite germplasm in the individual progeny of aparticular cross. For example, when a plant with a desired integrationevent which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the integration event of interestbut also have a relatively large proportion of the desired germplasm. Inthis way, the number of generations required to introgress one or moreinsertion events into a particular genetic background is minimized.

In the process of marker-assisted breeding, DNA sequences are used tofollow particular traits in the process of plant breeding (Tanksley etal., 1989). In terms of the present invention, such a desirable traitmay comprise, for example, a particular insertion event of a transgeneor an endogenous element such as a transposon. Marker-assisted breedingmay be undertaken as follows. Seeds of plants with the desired trait areplanted in soil in the greenhouse or in the field. Leaf tissue isharvested from the plants for preparation of DNA at any point in growthat which approximately one gram of leaf tissue can be removed from eachplant without compromising the viability of the plant. Genomic DNA isisolated using a procedure modified from Shure et al. (1983).Approximately one gram of leaf tissue from each seedling is lypholyzedovernight in 15 ml polypropylene tubes. Freeze-dried tissue is ground toa powder in the tubes using a glass rod. Powdered tissue is mixedthoroughly with 3 ml extraction buffer (7.0 urea, 0.35 M NaCl; 0.05 MTris-HCD, pH 8.0; 0.01 M EDTA; and 1% sarcosine). Tissue/bufferhomogenate is extracted with 3 ml phenol/chloroform. The aqueous phaseis separated by centrifugation and precipitated twice using 1/10 volumeof 4.4 M ammonium acetate (pH 5.2) and an equal volume of isopropanol.The precipitate is washed with 75% ethanol and resuspended in 100-500 μlTE (0.01 M Tris-HCl and 0.001 M EDTA, pH 8.0).

Genomic DNA is then digested with a 3-fold excess of restrictionenzymes, electrophoresed through 0.8% agarose (FMC), and transferred(Southern, 1975) to Nytran (Schleicher and Schuell) using 10×SCP(20×SCP=2 M NaCl, 0.6 M disodium phosphate and 0.02 M disodium EDTA).The filters are prehybridized in 6×SCP, 10% dextran sulfate, 2%sarcosine, 500 μg/ml denatured salmon sperm DNA, and ³²P-labeled probegenerated by random priming (Feinberg & Vogelstein, 1983). Hybridizedfilters are washed in 2×SCP and 1% SDS at 65° C. for 30 minutes andvisualized by autoradiography using Kodak XAR5 film. Geneticpolymorphisms which are genetically linked to traits of interest arethereby used to predict the presence or absence of the traits ofinterest.

Those of skill in the art will recognize that there are many differentways to isolate DNA from plant tissues and that there are many differentprotocols for Southern hybridization that will produce identicalresults. Those of skill in the art will recognize that a Southern blotcan be stripped of radioactive probe following autoradiography andre-probed with a different probe. In this manner. one may identify eachof the various integration events that are present in the plant.Further, one of skill in the art will recognize that any type of geneticmarker which is polymorphic at the region(s) of interest may be used forthe purpose of identifying the relative presence or absence of a traitand that such information may be used for marker assisted breeding.

Each lane of a Southern blot represents DNA isolated from one plant.Through the use of multiplicity of gene integration events as probes onthe same genomic DNA blot, the integration event composition of eachplant may be determined. Correlations may be established between thecontributions of particular integration events to the phenotype of theplant. Only those plants that contain a desired combination ofintegration events may be desired for advancement to maturity and usefor pollination. DNA probes corresponding to particular integrationevents are useful markers during the course of plant breeding toidentify and combine particular integration events without having togrow the plants and assay the plants for agronomic performance.

It is expected that one or more restriction enzymes will be used todigest genomic DNA, either singly or in combinations. One of skill inthe art will recognize that many different restriction enzymes will beuseful, and the choice of restriction enzyme will depend on the DNAsequence of the transgene integration event that is used as a probe andthe DNA sequences in the genome surrounding the transgene. For a probe,one will want to use DNA or RNA sequences which will hybridize to theDNA used for transformation. One will select a restriction enzyme thatproduces a DNA fragment following hybridization that is identifiable asthe transgene integration event. Thus, particularly useful restrictionenzymes will be those which reveal polymorphisms that are geneticallylinked to specific transgenes or traits of interest.

EXAMPLE 17 Utilization of Insertionally Mutated Crops

One embodiment of the current invention has, as an ultimate goal, theproduction of novel plants which will be useful to man. Such plants maycomprise a transformation event having a selected site of integration,may comprise in their genomes a desired insertion mutation, or may betransformed with one or more genes the function of which has beendetermined with the current invention. It is specifically contemplatedby the inventor that such plants may be used for virtually any purposedeemed of value. For example, one may wish to harvest seed from plantswith a particular insertion event or transgene. This seed may in turn beused for a wide variety of purposes. The seed may be sold to farmers forplanting in the field or may be directly used as food, either foranimals or humans. Alternatively, products may be made from the seeditself. Examples of products which may be made from the seed include,oil, starch, animal or human food, pharmaceuticals, and variousindustrial products. Such products may be made from particular plantparts or from the entire plant. One product made from the entire plantwhich is deemed of particular value is silage for animal feed.

Means for preparing products from plants, such as those that may beidentified with the current invention, have been well known since thedawn of agriculture and will be known to those of skill in the art inlight of the instant disclosure. Specific methods for crop utilizationmay be found in, for example, Sprague et al. (1988), and Watson et al.(1987).

EXAMPLE 18 General Methods for Assays

DNA analysis is performed as follows. Genomic DNA is isolated using aprocedure modified from Shure et al. (1983). Approximately 1 gm tissueis ground to a fine powder in liquid nitrogen using a mortar and pestle.Powdered tissue is mixed thoroughly with 4 ml extraction buffer (7.0 Murea; 0.35 M NaCl; 0.05 M Tris-HCl, pH 8.0; 0.01 M EDTA; and 1%sarcosine). Tissue/buffer homogenate is extracted with 4 mlphenol/chloroform. The aqueous phase is separated by centrifugation,passed through Miracloth, and precipitated twice using 1/10 volume of4.4 M ammonium acetate (pH 5.2) and an equal volume of isopropanol. Theprecipitate is washed with 70% ethanol and resuspended in 200-500:1 TE(0.01 M Tris-Hcl and 0.001 M EDTA, pH 8.0).

The presence of a particular sequence in a plant may be detected throughthe use of polymerase chain reaction (PCR). Using this technique,specific fragments of DNA can be amplified and detected followingagarose gel electrophoresis. For example, two hundred to 1000 ng genomicDNA is added to a reaction mix containing 10 mM Tris-HCl (pH 8.3); 1.5mM MgCl₂; 50 mM KCl; 0.1 mg/ml gelatin; 200 μM each dATP, dCTP, dGTP,and dTTP; 0.5 μM each forward and reverse DNA primers; 20% glycerol; and2.5 U Taq DNA polymerase. The reaction is run in a thermal cyclingmachine as follows with 39 repeats of the cycle: 94° C. for 3 min, 94°C. for 1 min, 50° C. for 1 min, and 72° C. for 30 s, followed by 72° C.for 5 min. Twenty μl of each reaction mix is run on a 3.5% NuSieve gelin TBE buffer (90 mM Tris-borate and 2 mM EDTA) at 50V for two to fourhours. Using this procedure, for example, one may detect the presence ofa bar gene integration event using the forward primerCATCGAGACAAGCACGGTCAACTTC (SEQ ID NO:8) and the reverse primerAAGTCCCTGGAGGCACAGGGCTTCAAGA. (SEQ ID NO:9)

For Southern blot analysis, genomic DNA is digested with a 3-fold excessof restriction enzymes, electrophoresed through 0.8% agarose (FMC), andtransferred (Southern, 1975) to Nytran (Schleicher and Schuell) using10×SCP (20×SCP=2 M NaCl, 0.6 M disodium phosphate, and 0.02 M disodiumEDTA). Filters are prehybridized at 65° C. in 6×SCP, 10% dextransulfate, 2% sarcosine, and 500 μg/ml heparin (Chomet et aL, 1987) for 15min. Filters then are hybridized overnight at 65° C. in 6×SCP containing100 μg/ml denatured salmon sperm DNA and ³²P-labeled probe. Filters arewashed in 2×SCP and 1% SDS at 65° C. for 30 min and visualized byautoradiography using Kodak XAR5 film. For rehybridization, the filtersare boiled for 10 min in distilled H₂O to remove the first probe andthen prehybridized as described above.

EXAMPLE 19 Selection of Desirable Transformation Events

It is specifically contemplated by the inventor that the currentinvention may be used to select for transformation events which arelocated in a particular region of a genome. This is significant, becausethe genomic location of a transformation event will greatly influencethe expression of a transgene. Therefore, one may determine regions ofthe genome in which a transgene will be highly expressed, clone DNA fromthat region, and then use that clone to select transformation eventsfrom the region of interest using that probe.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe methods of this invention have been described in terms of thepreferred embodiments, it will be apparent to those of skill in the artthat variations may be applied to the methods and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit, and scope of the invention. More specifically, itwill be apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while achieving the same or similar results. All such similarsubstitutes and modifications apparent to those skilled in the art aredeemed to be within the concept, spirit, and scope of the invention asdefined by the appended claims.

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9 1 6743 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 1 agtactttga tccaacccct ccgctgctat agtgcagtcggcttctgacg ttcagtgcag 60 ccgtcttctg aaaacgacat gtcgcacaag tcctaagttacgcgacaggc tgccgccctg 120 cccttttcct ggcgttttct tgtcgcgtgt tttagtcgcataaagtagaa tacttgcgac 180 tagaaccgga gacattacgc catgaacaag agcgccgccgctggcctgct gggctatgcc 240 cgcgtcagca ccgacgacca ggacttgacc aaccaacgggccgaactgca cgcggccggc 300 tgcaccaagc tgttttccga gaagatcacc ggcaccaggcgcgaccgccc ggagctggcc 360 aggatgcttg accacctacg ccctggcgac gttgtgacagtgaccaggct agaccgcctg 420 gcccgcagca cccgcgacct actggacatt gccgagcgcatccaggaggc cggcgcgggc 480 ctgcgtagcc tggcagagcc gtgggccgac accaccacgccggccggccg catggtgttg 540 accgtgttcg ccggcattgc cgagttcgag cgttccctaatcatcgaccg cacccggagc 600 gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcccccgccctac cctcaccccg 660 gcacagatcg cgcacgcccg cgagctgatc gaccaggaaggccgcaccgt gaaagaggcg 720 gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcgcacttgagcg cagcgaggaa 780 gtgacgccca ccgaggccag gcggcgcggt gccttccgtgaggacgcatt gaccgaggcc 840 gacgccctgg cggccgccga gaatgaacgc caagaggaacaagcatgaaa ccgcaccagg 900 acggccagga cgaaccgttt ttcattaccg aagagatcgaggcggagatg atcgcggccg 960 ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcggctgcatgaa atcctggccg 1020 gtttgtctga tgccaagctg gcggcctggc cggccagcttggccgctgaa gaaaccgagc 1080 gccgccgtct aaaaaggtga tgtgtatttg agtaaaacagcttgcgtcat gcggtcgctg 1140 cgtatatgat gcgatgagta aataaacaaa tacgcaaggggaacgcatga aggttatcgc 1200 tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatcgcaacccatc tagcccgcgc 1260 cctgcaactc gccggggccg atgttctgtt agtcgattccgatccccagg gcagtgcccg 1320 cgattgggcg gccgtgcggg aagatcaacc gctaaccgttgtcggcatcg accgcccgac 1380 gattgaccgc gacgtgaagg ccatcggccg gcgcgacttcgtagtgatcg acggagcgcc 1440 ccaggcggcg gacttggctg tgtccgcgat caaggcagccgacttcgtgc tgattccggt 1500 gcagccaagc ccttacgaca tatgggccac cgccgacctggtggagctgg ttaagcagcg 1560 cattgaggtc acggatggaa ggctacaagc ggcctttgtcgtgtcgcggg cgatcaaagg 1620 cacgcgcatc ggcggtgagg ttgccgaggc gctggccgggtacgagctgc ccattcttga 1680 gtcccgtatc acgcagcgcg tgagctaccc aggcactgccgccgccggca caaccgttct 1740 tgaatcagaa cccgagggcg acgctgcccg cgaggtccaggcgctggccg ctgaaattaa 1800 atcaaaactc atttgagtta atgaggtaaa gagaaaatgagcaaaagcac aaacacgcta 1860 agtgccggcc gtccgagcgc acgcagcagc aaggctgcaacgttggccag cctggcagac 1920 acgccagcca tgaagcgggt caactttcag ttgccggcggaggatcacac caagctgaag 1980 atgtacgcgg tacgccaagg caagaccatt accgagctgctatctgaata catcgcgcag 2040 ctaccagagt aaatgagcaa atgaataaat gagtagatgaattttagcgg ctaaaggagg 2100 cggcatggaa aatcaagaac aaccaggcac cgacgccgtggaatgcccca tgtgtggagg 2160 aacgggcggt tggccaggcg taagcggctg ggttgtctgccggccctgca atggcactgg 2220 aacccccaag cccgaggaat cggcgtgacg gtcgcaaaccatccggcccg gtacaaatcg 2280 gcgcggcgct gggtgatgac ctggtggaga agttgaaggccgcgcaggcc gcccagcggc 2340 aacgcatcga ggcagaagca cgccccggtg aatcgtggcaagcggccgct gatcgaatcc 2400 gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtcgattaggaag ccgcccaagg 2460 gcgacgagca accagatttt ttcgttccga tgctctatgacgtgggcacc cgcgatagtc 2520 gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcgtgaccgacga gctggcgagg 2580 tgatccgcta cgagcttcca gacgggcacg tagaggtttccgcagggccg gccggcatgg 2640 ccagtgtgtg ggattacgac ctggtactga tggcggtttcccatctaacc gaatccatga 2700 accgataccg ggaagggaag ggagacaagc ccggccgcgtgttccgtcca cacgttgcgg 2760 acgtactcaa gttctgccgg cgagccgatg gcggaaagcagaaagacgac ctggtagaaa 2820 cctgcattcg gttaaacacc acgcacgttg ccatgcagcgtacgaagaag gccaagaacg 2880 gccgcctggt gacggtatcc gagggtgaag ccttgattagccgctacaag atcgtaaaga 2940 gcgaaaccgg gcggccggag tacatcgaga tcgagctagctgattggatg taccgcgaga 3000 tcacagaagg caagaacccg gacgtgctga cggttcaccccgattacttt ttgatcgatc 3060 ccggcatcgg ccgttttctc taccgcctgg cacgccgcgccgcaggcaag gcagaagcca 3120 gatggttgtt caagacgatc tacgaacgca gtggcagcgccggagagttc aagaagttct 3180 gtttcaccgt gcgcaagctg atcgggtcaa atgacctgccggagtacgat ttgaaggagg 3240 aggcggggca ggctggcccg atcctagtca tgcgctaccgcaacctgatc gagggcgaag 3300 catccgccgg ttcctaatgt acggagcaga tgctagggcaaattgcccta gcaggggaaa 3360 aaggtcgaaa aggtctcttt cctgtggata gcacgtacattgggaaccca aagccgtaca 3420 ttgggaaccg gaacccgtac attgggaacc caaagccgtacattgggaac cggtcacaca 3480 tgtaagtgac tgatataaaa gagaaaaaag gcgatttttccgcctaaaac tctttaaaac 3540 ttattaaaac tcttaaaacc cgcctggcct gtgcataactgtctggccag cgcacagccg 3600 aagagctgca aaaagcgcct acccttcggt cgctgcgctccctacgcccc gccgcttcgc 3660 gtcggcctat cgcggccgct ggccgctcaa aaatggctggcctacggcca ggcaatctac 3720 cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccggcgcccacat caaggcaccc 3780 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgacacatgcagct cccggagacg 3840 gtcacagctt gtctgtaagc ggatgccggg agcagacaagcccgtcaggg cgcgtcagcg 3900 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcacgtagcgatag cggagtgtat 3960 actggcttaa ctatgcggca tcagagcaga ttgtactgagagtgcaccat atgcggtgtg 4020 aaataccgca cagatgcgta aggagaaaat accgcatcaggcgctcttcc gcttcctcgc 4080 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagcggtatcagct cactcaaagg 4140 cggtaatacg gttatccaca gaatcagggg ataacgcaggaaagaacatg tgagcaaaag 4200 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgctggcgtttttc cataggctcc 4260 gcccccctga cgagcatcac aaaaatcgac gctcaagtcagaggtggcga aacccgacag 4320 gactataaag ataccaggcg tttccccctg gaagctccctcgtgcgctct cctgttccga 4380 ccctgccgct taccggatac ctgtccgcct ttctcccttcgggaagcgtg gcgctttctc 4440 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgttcgctccaag ctgggctgtg 4500 tgcacgaacc ccccgttcag cccgaccgct gcgccttatccggtaactat cgtcttgagt 4560 ccaacccggt aagacacgac ttatcgccac tggcagcagccactggtaac aggattagca 4620 gagcgaggta tgtaggcggt gctacagagt tcttgaagtggtggcctaac tacggctaca 4680 ctagaaggac agtatttggt atctgcgctc tgctgaagccagttaccttc ggaaaaagag 4740 ttggtagctc ttgatccggc aaacaaacca ccgctggtagcggtggtttt tttgtttgca 4800 agcagcagat tacgcgcaga aaaaaaggat ctcaagaagatcctttgatc ttttctacgg 4860 ggtctgacgc tcagtggaac gaaaactcac gttaagggattttggtcatg catgatatat 4920 ctcccaattt gtgtagggct tattatgcac gcttaaaaataataaaagca gacttgacct 4980 gatagtttgg ctgtgagcaa ttatgtgctt agtgcatctaatcgcttgag ttaacgccgg 5040 cgaagcggcg tcggcttgaa cgaatttcta gctagacattatttgccgac taccttggtg 5100 atctcgcctt tcacgtagtg gacaaattct tccaactgatctgcgcgcga ggccaagcga 5160 tcttcttctt gtccaagata agcctgtcta gcttcaagtatgacgggctg atactgggcc 5220 ggcaggcgct ccattgccca gtcggcagcg acatccttcggcgcgatttt gccggttact 5280 gcgctgtacc aaatgcggga caacgtaagc actacatttcgctcatcgcc agcccagtcg 5340 ggcggcgagt tccatagcgt taaggtttca tttagcgcctcaaatagatc ctgttcagga 5400 accggatcaa agagttcctc cgccgctgga cctaccaaggcaacgctatg ttctcttgct 5460 tttgtcagca agatagccag atcaatgtcg atcgtggctggctcgaagat acctgcaaga 5520 atgtcattgc gctgccattc tccaaattgc agttcgcgcttagctggata acgccacgga 5580 atgatgtcgt cgtgcacaac aatggtgact tctacagcgcggagaatctc gctctctcca 5640 ggggaagccg aagtttccaa aaggtcgttg atcaaagctcgccgcgttgt ttcatcaagc 5700 cttacggtca ccgtaaccag caaatcaata tcactgtgtggcttcaggcc gccatccact 5760 gcggagccgt acaaatgtac ggccagcaac gtcggttcgagatggcgctc gatgacgcca 5820 actacctctg atagttgagt cgatacttcg gcgatcaccgcttcccccat gatgtttaac 5880 tttgttttag ggcgactgcc ctgctgcgta acatcgttgctgctccataa catcaaacat 5940 cgacccacgg cgtaacgcgc ttgctgcttg gatgcccgaggcatagactg taccccaaaa 6000 aaacatgtca taacaagaag ccatgaaaac cgccactgcgccgttaccac cgctgcgttc 6060 ggtcaaggtt ctggaccagt tgcgtgacgg cagttacgctacttgcatta cagcttacga 6120 accgaacgag gcttatgtcc actgggttcg tgcccgaattgatcacaggc agcaacgctc 6180 tgtcatcgtt acaatcaaca tgctaccctc cgcgagatcatccgtgtttc aaacccggca 6240 gcttagttgc cgttcttccg aatagcatcg gtaacatgagcaaagtctgc cgccttacaa 6300 cggctctccc gctgacgccg tcccggactg atgggctgcctgtatcgagt ggtgattttg 6360 tgccgagctg ccggtcgggg agctgttggc tggctggtggcaggatatat tgtggtgtaa 6420 acaaattgac gcttagacaa cttaataaca cattgcggacgtttttaatg tactgaatta 6480 acgccgaatt gaattcgagc tcggtacccg gggatcctctagagtcgacc tgcaggcatg 6540 caagcttagc ttgagcttgg atcagattgt cgtttcccgccttcagttta aactatcagt 6600 gtttgacagg atatattggc gggtaaacct aagagaaaagagcgtttatt agaataacgg 6660 atatttaaaa gggcgtgaaa aggtttatcc gttcgtccatttgtatgtgc atgccaacca 6720 cagggttccc ctcgggatca aac 6743 2 14 DNA Zeamays 2 tactcaggac tcat 14 3 16 DNA Zea mays 3 gacgatgagt cctgag 16 4 28DNA Zea mays 4 agagaagcca acgccaatcg cctccatt 28 5 20 DNA Zea mays 5gatgagtcct gagtaaagct 20 6 20 DNA Zea mays 6 gatgagtcct gagtagagct 20 720 DNA Zea mays 7 tctataatgg caattatctc 20 8 25 DNA Streptomyceshygroscopicus 8 catcgagaca agcacggtca acttc 25 9 28 DNA Streptomyceshygroscopicus 9 aagtccctgg aggcacaggg cttcaaga 28

What is claimed is:
 1. A method of identifying an insertion event in atarget locus comprising the steps of: (a) preparing a first DNAcomposition enhanced for a plurality of insertion junctions; (b)creating a pooled DNA composition comprising DNA from said first DNAcomposition and from at least a second DNA composition enhanced for aplurality of insertion junctions; (c) labeling said pooled DNAcomposition; (d) preparing at least a first detectable array comprisingnucleic acid sequences from a set of target loci, wherein said preparingcomprises directly or indirectly attaching the nucleic acids from saidset of target loci to a solid support; and (e) hybridizing said labeledpooled DNA composition to said array to identify at least a first targetlocus which comprises an insertion event.
 2. The method of claim 1,wherein said step of preparing said DNA composition comprisesamplification of insertion junctions with inverse PCR.
 3. The method ofclaim 1, wherein said step of preparing said DNA composition comprisesamplification of insertion junctions with vectorette PCR.
 4. The methodof claim 1, wherein said step of preparing said DNA compositioncomprises amplification of insertion junctions with primer-adapted PCR.5. The method of claim 1, wherein said step of preparing said DNAcomposition comprises amplification of insertion junctions with AIMS. 6.The method of claim 1, wherein said nucleic acids from a set of targetloci comprise DNA.
 7. The method of claim 1, wherein said solid supportcomprises a microscope slide.
 8. The method of claim 7, wherein saidstep of labeling comprises labeling said DNA composition with a visuallydetectable marker.
 9. The method of claim 1, wherein said labelingcomprises labeling said DNA composition with an antigen, and whereinsaid antigen is detected with a molecule which binds said antigen. 10.The method of claim 1, wherein said solid support comprises ahybridization filter.
 11. The method of claim 1, wherein said labelingcomprises labeling said DNA composition with a radiolabel.
 12. Themethod of claim 11, wherein said radiolabel is detected byautoradiography.
 13. The method of claim 1, wherein said DNA compositioncomprises a pool.
 14. The method of claim 1, wherein said DNAcomposition comprises plant DNA.
 15. The method of claim 14, whereinsaid plant DNA is further defined as monocot plant DNA.
 16. The methodof claim 15, wherein said monocot plant DNA is still further defined asderived from a species selected from the group consisting of maize,rice, wheat, barley, sorghum, oat, and sugarcane.
 17. The method ofclaim 16, wherein said monocot DNA is maize DNA.
 18. The method of claim14, wherein said plant DNA is further defined as dicot plant DNA. 19.The method of claim 18, wherein said dicot DNA is further defined asderived from a species selected from the group consisting of cotton,tobacco, tomato, soybean, sunflower, oil seed rape (canola), alfalfa,potato, strawberry, onion, broccoli, Arabidopsis, pepper, and citrus.20. The method of claim 19, wherein said dicot DNA is still furtherdefined as comprising Arabidopsis thaliana DNA.
 21. The method of claim1, wherein said DNA composition comprises animal DNA.
 22. The method ofclaim 8, wherein said visually detectable marker comprises afluorophore.
 23. The method of claim 8, wherein said visually detectablemarker comprises a chromophore.
 24. The method of claim 8, wherein saidvisually detectable marker comprises a chemiluminescent moiety.
 25. Themethod of claim 1, wherein said labeling comprises labeling said DNAcomposition with a transition metal.
 26. The method of claim 6, whereinsaid nucleic acids from a set of target loci comprise DNA from thecoding sequence of a target organism.
 27. The method of claim 26,wherein the coding sequence DNA comprises cDNA.
 28. The method of claim27, wherein said coding sequence DNA encodes a functional polypeptide.29. The method of claim 1, wherein said DNA from a set of target locicomprises genomic DNA.
 30. The method of claim 1, wherein said nucleicacids from a set of target loci comprise plant DNA.
 31. The method ofclaim 30, wherein said plant DNA is further defined as monocot plantDNA.
 32. The method of claim 31, wherein said monocot plant DNA is stillfurther defined as derived from a species selected from the groupconsisting of maize, rice, wheat, barley, sorghum, oat, and sugarcane.33. The method of claim 32, wherein said monocot plant DNA is maize DNA.34. The method of claim 30, wherein said plant DNA is further defined asdicot plant DNA.
 35. The method of claim 34, wherein said dicot plantDNA is further defined as derived from a species selected from the groupconsisting of cotton, tobacco, tomato, soybean, sunflower, oil seed rape(canola), alfalfa, potato, strawberry, onion, broccoli, Arabidopsis,pepper, and citrus.
 36. The method of claim 35, wherein said dicot plantDNA is Arabidopsis thaliana DNA.
 37. The method of claim 1, wherein saidnucleic acids from a set of target loci comprise animal DNA.
 38. Themethod of claim 1, wherein said insertion junctions are cloned prior tosaid labeling.
 39. A method of identifying an insertion event in atarget locus comprising the steps of: (a) preparing a first DNAcomposition enhanced for a plurality of insertion junctions; (b)creating a pooled DNA composition comprising DNA from said first DNAcomposition and from at least a second DNA composition enhanced for aplurality of insertion junctions; (c) sequencing said insertionjunctions in said pooled DNA composition; and (d) identifying aninsertion event in a target locus by screening the sequenced insertionjunctions for a nucleic acid sequence corresponding to said targetlocus.
 40. The method of claim 39, wherein said DNA composition enhancedfor a plurality of insertion junctions comprises a pool.
 41. The methodof claim 39, wherein said screening comprises conducting a homologysearch.
 42. The method of claim 39, wherein said step of preparing saidDNA composition comprises amplification of insertion junctions withinverse PCR.
 43. The method of claim 39, wherein said step of preparingsaid DNA composition comprises amplification of insertion junctions withvectorette PCR.
 44. The method of claim 39, wherein said step ofpreparing said DNA composition comprises amplification of insertionjunctions with primer-adapted PCR.
 45. The method of claim 39, whereinsaid step of preparing said DNA composition comprises amplification ofinsertion junctions with AIMS.
 46. The method of claim 39, wherein saidDNA composition comprises plant DNA.
 47. The method of claim 46, whereinsaid plant DNA is monocot plant DNA.
 48. The method of claim 47, whereinsaid monocot plant DNA is still further defined as derived from aspecies selected from the group consisting of maize, rice, wheat,barley, sorghum, oat, and sugarcane.
 49. The method of claim 48, whereinsaid monocot plant DNA is maize DNA.
 50. The method of claim 46, whereinsaid plant DNA is dicot plant DNA.
 51. The method of claim 50, whereinsaid dicot plant DNA is further defined as derived from a speciesselected from the group consisting of cotton, tobacco, tomato, soybean,sunflower, oil seed rape (canola), alfalfa, potato, strawberry, onion,broccoli, Arabidopsis, pepper, and citrus.
 52. The method of claim 51,wherein said dicot plant DNA is Arabidopsis thaliana DNA.
 53. The methodof claim 39, wherein said DNA composition comprises animal DNA.
 54. Themethod of claim 39, wherein said target locus is from a plant.
 55. Themethod of claim 54, wherein said plant is a monocot plant.
 56. Themethod of claim 55, wherein said monocot plant is selected from thegroup consisting of maize, rice, wheat, barley, sorghum, oat, andsugarcane.
 57. The method of claim 56, wherein said monocot plant ismaize.
 58. The method of claim 54, wherein said plant is a dicot plant.59. The method of claim 58, wherein said dicot plant is selected fromthe group consisting of cotton, tobacco, tomato, soybean, sunflower, oilseed rape (canola), alfalfa, potato, strawberry, onion, broccoli,Arabidopsis, pepper, and citrus.
 60. The method of claim 59, whereinsaid dicot plant is Arabidopsis thaliana.
 61. The method of claim 39,wherein said target locus is from an animal.